Methods, devices, and compositions for lysis of occlusive blood clots while sparing wound sealing clots

ABSTRACT

It has now been discovered that certain mutant forms of pro-urokinase (“pro-UK”), such as so-called pro-UK mutant “M5” (Lys.sup.300.fwdarw.His)-, perform in the manner of pro-UK in lysing “bad” blood clots (those clots that occlude blood vessels), while sparing hemostatic fibrin in the so-called “good” blood clots (those clots that seal wounds, e.g., after surgery or other tissue injury). Thus, these pro-UK mutants are excellent and safe thrombolytic agents. These advantages allow them to be used in a variety of new methods, devices, and compositions useful for thrombolysis and treating various cardiovascular disorders in clinical situations where administration of other known thrombolytic agents has been too risky or even contraindicated.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional PatentApplication Ser. Nos. 60/464,003, 60/463,930, and 60/464,002, all filedon Apr. 18, 2003. The contents of each of these applications areincorporated herein by reference in their entirety.

TECHNICAL FIELD

The invention relates to the use of prourokinase mutants in variousmethods, devices, and compositions for therapeutic thrombolysis withoutinducing hemorrhage.

BACKGROUND

The leading two causes of death listed by the World Health Organization(1998) are coronary heart disease and cerebrovascular disease. Sincethese diseases are largely triggered by blood clots, there is aconsiderable need for safe and effective thrombolytic agents (drugscapable of dissolving clots and restoring blood flow). However, bloodclots also perform the essential physiological function of preventinghemorrhage by sealing injured vessels. This process is called hemostasisand most thrombolytic drugs interfere with hemostasis by inducing ahemophilia-like state. More importantly, these drugs lyse hemostaticfibrin (which seals injuries). By these two mechanism, thrombolytictherapy has carried significant hemorrhagic risk.

When intravascular blood clotting occurs an occlusive clot or thrombusforms, and blood flow is often arrested at that site. Blood clotsconsist largely of fibrin, which is a natural polymer that forms fromfibrinogen in blood as the end-product of clotting. Depending on thelocation in the arterial system, i.e., heart, brain, or leg, such a clotcan trigger a heart attack, stroke, or peripheral gangrene. In thevenous circulation the same process can cause thrombophlebitis (deepvein thrombosis) or pulmonary embolism (lung clots). Together, thesecardiovascular diseases constitute the leading causes of death anddisability in industrialized countries. Since the tendency to formocclusive clots increases with age and populations are getting older,the incidence of these disorders is increasing worldwide.

Not surprisingly, blood clotting and thrombolysis have been a majorfocus of biomedical research over the past 30 years, and this researchhas produced an array of anti-clotting (anticoagulant) as well asclot-dissolving (thrombolytic) drugs. The first thrombolytic drugs to bedeveloped were streptokinase (SK) and urokinase (UK), both of which havecertain shortcomings, e.g., SK is antigenic and has limited efficacy,and both SK and UK induce non-specific effects, since they do not targetblood clots, and act systemically upon constituents of healthy blood,causing the hemophilia-like state referred to above.

Tissue plasminogen activator (t-PA) was developed some years later andwas one of the first biotechnology products. T-PA is non-antigenic,since it is a natural enzyme, is clot-specific (less likely to cause thehemophilia-like state), and is almost twice as effective as SK in lysingblood clots in vitro. However, when t-PA was tested clinically, it wasfound to induce more hemorrhagic side effects, and be associated with ahigher stroke and reocclusion rate than SK, despite its superiorspecificity. These and other side effects caused its clinical benefitsin the treatment of heart attacks to be little better than thoseobtained with SK.

Another thrombolytic agent, pro-urokinase (pro-UK) is a natural zymogenthat activates plasminogen, to form plasmin, which in turn activatespro-UK to UK. Like t-PA, pro-UK is known to be selective for plasminogenbound to blood clots (see, e.g., Husain et al., U.S. Pat. No.4,381,346), in contrast to UK (or SK), which activates plasminogenindiscriminately. This is a problem because of the high concentration ofplasminogen in blood. Thus, pro-UK is referred to as fibrinclot-specific, or selective, whereas UK is non-specific. Pro-UK seemedbetter adapted to pharmacological use than SK, UK, or t-PA, because itis substantially inert in the blood (being a pro-enzyme) atphysiological concentrations. Its activation is dependent on thepresence of a fibrin clot or thrombus. Unfortunately, at therapeuticdoses, which are significantly larger than naturally occurringconcentrations, pro-UK becomes unstable and is readily converted byplasmin to UK. When this occurs, the selective mechanism of action ofpro-UK is lost, and the hemophilia-like side effects and bleeding takeplace.

Because of such shortcomings of presently available thrombolytic agents,heart attacks are currently treated with angioplasty and stents, despitetheir technical complexity, cost, and associated delay in treatment ofthe patient. Out of the 1 to 2 million heart attacks that occur in theU.S. and Europe annually, a growing percentage is being treated by theseinvasive procedures. This is because a number of studies havedemonstrated that the clinical outcome is better than with thecommercially available thrombolytic drugs, and there is no risk ofbleeding or hemorrhagic stroke.

The only new thrombolytic drugs that have appeared on the market in thepast five years are mutant forms of t-PA. These mutant t-PAs have anefficacy and side effects essentially identical to those of t-PA, butcan be administered as a bolus injection rather than by an extendedinfusion. Like t-PA and SK, these drugs are inimical to angioplasty,which, when possible, has become the treatment of choice for most heartattacks. Therefore, coronary reperfusion is delayed until a patient isbrought into an adequately staffed catheterization laboratory. Thistakes at least 60-90 minutes, a critical time during which significantheart muscle is permanently lost due to lack of blood supply(perfusion).

Mutant forms of pro-UK are described in Liu et al., U.S. Pat. No.5,472,692. These pro-UK mutants are said to have lower intrinsicactivity than pro-UK and are more stable in plasma than native pro-UK.The pro-UK mutants are said to be used and administered as thrombolyticagents.

SUMMARY

The invention is based, at least in part, on the discovery that certainmutant forms of pro-UK (“pro-UK mutants”) such as pro-UK mutant “M5” (asdefined herein), are plasminogen activators that spare “good” fibrinclots (the hemostatic fibrin that seals injured blood vessels), while atthe same time lysing the “bad” clots (that occlude blood vessels). Thehemostatic fibrin is also important for repair of spontaneous vesselinjury, a particularly common event in the elderly. The invention isalso based in part on the realization of the importance, in thisconnection, that the substrate for pro-UK and pro-UK mutants,plasminogen, has different conformations depending on whether it isbound to fibrin in an occlusive blood clot or a wound sealing bloodclot. It is believed that this difference in conformation of plasminogenallows the pro-UK mutants to preferentially lyse the bad clots and sparethe good clots. Furthermore, certain mutants such as M5, have abouttwice the fibrinolytic activity in a plasma milieu at fibrin-specificconcentrations as pro-UK (or t-PA).

In general, in one aspect, the invention features methods of treating aperson with symptoms of stroke by (a) determining that a personpotentially has had a stroke based on symptoms of stroke, e.g., withoutthe need for medical confirmation or a complete diagnosis; and (b)administering to the person a composition comprising an amount of apro-UK mutant, e.g., a so-called “flexible loop” mutant such as M5,effective to lyse any potential blood clot causing the symptoms ofstroke. In these methods, the composition can be administered more than3 hours after the onset of symptoms, a bolus of the composition can beadministered including 20-50 mg of the pro-UK mutant, and the methodscan further include obtaining a medical confirmation of an occlusivethrombus in the brain (e.g., by CT scan), and administering an infusionof the composition at a pro-UK flexible loop mutant dosage of dose of120-200 mg/hour (intravenous) or 50-100 mg/hour (intra-arterial).

A pro-UK flexible loop mutant is a polypeptide that has the amino acidsequence of wild-type pro-UK (which has 411 amino acids), but with oneor more amino acids in the “flexible loop” (which includes the aminoacids at locations 297-313) replaced by a neutral amino acid such asalanine (Ala) or an amino acid that can take on only a weak positivecharge, such as histidine (His). These flexible loop mutants aredescribed in U.S. Pat. No. 5,472,692. One example of a pro-UK flexibleloop mutant is M5, which has the complete amino acid sequence ofwild-type pro-UK, but with one amino acid alteration, Lys³⁰⁰→His.

Pro-UK flexible loop mutants “spare” wound sealing blood clots, whichmeans that they cause lysis (via plasminogen) of fibrin in occlusiveblood clots preferentially to the fibrin in wound sealing clots.

“Medical confirmation,” refers to confirmation of an initial diagnosisbased on a patient's symptoms (either observed by the physician or EMT,or described by the patient), by medical testing such as with medicaldevices, blood test, and the like.

In another aspect, the invention features methods of treating a personwith symptoms of a heart attack by (a) diagnosing a patient aspotentially having a heart attack based on symptoms of a heart attack,e.g., without the need for medical confirmation; and (b) administeringto the potential heart attack patient a composition comprising an amountof a pro-urokinase mutant, e.g., a flexible loop mutant such as M5,effective to lyse any potential blood clot causing the symptoms of aheart attack. In these methods, the composition can be administeredwithin 90 minutes of the onset of symptoms, and a bolus of thecomposition can be administered including 20-50 mg of the pro-UK mutant.In addition, the methods can further include obtaining a medicalconfirmation of an occlusive thrombus in a coronary artery, andadministering an infusion of the composition at a pro-UK mutant dosageof 50-200 mg/hour.

As used herein, the term “diagnosing” refers to any observation ofsymptoms, such as symptoms of a potential stroke or heart attack, by anymedical personnel, including doctors, nurses, emergency medicaltechnicians (“EMTs”), physician's assistants, and the like, or even alay person with some minimal medical training. The term “diagnosing”does not require a formal diagnosis made by a doctor, and the diagnosiscan be made in the field, e.g., in the patient's home or other publicplace, or in a doctor's office or hospital.

In other embodiments, the invention features methods of lysing occlusivethrombi and emboli in a patient before, during, or after surgery, byadministering to the patient a composition comprising an amount of apro-UK mutant, e.g., a flexible loop mutant such as M5, effective topreferentially lyse any potential occlusive thrombus or embolus comparedto hemostatic fibrin in wound sealing clots. In this method, thecomposition can be administered by infusion within about one, two, orthree hours before or after (up to 24 hours after) surgery, or duringsurgery, and the composition can be administered by infusion at a pro-UKflexible loop mutant dosage of 50-200 mg/hour, or performing balloonangioplasty.

The invention also includes an intravascular expandable catheter fordelivering to a vascular site in a patient an activated, two-chainpro-UK mutant, e.g., flexible loop mutant, (“mutant UK”). The catheterincludes (a) a catheter body having proximal and distal ends; (b) anexpandable portion arranged at the distal end of the catheter body; and(c) a carrier layer arranged on a surface of the expandable portion,wherein the carrier layer includes an amount of an activated pro-UKflexible loop mutant (“mutant UK”) effective to lyse thrombi or emboliin contact with the expandable portion. In this catheter, the carrierlayer can be a hydrogel selected to quickly release effective amounts ofthe mutant UK upon contact with a thrombus or embolus. The amount ofmutant UK can be, e.g., 0.1-0.5 mg. The carrier layer can include alumen containing the mutant UK and one or more apertures that arepressed against a thrombus or embolus to allow the thrombus or embolusto protrude into the one or more apertures, thereby contacting themutant UK.

In certain embodiments, the carrier layer can be two-chain pro-UK mutantM5, e.g., low molecular weight two-chain M5, and the expandable portioncan be an angioplasty balloon, or a stent-placement balloon.

In another aspect, the invention features an intravascular device, suchas a stent or suture, for delivering to a vascular site in a patient apro-urokinase (“pro-UK”) flexible loop mutant (“mutant UK”) thatincludes (a) a body; and (b) a carrier layer arranged on a surface ofthe body, wherein the carrier layer includes a sustained release agentthat slowly releases over time an amount of a pro-UK flexible loopmutant effective to lyse thrombi or emboli in contact with the body.

The invention also features methods of clearing lumens of blood clots byobtaining a lumen that contains or may contain a blood clot; and flowingthrough the lumen a solution comprising an activated, two-chain pro-UKmutant (“mutant UK”), e.g., activated, two-chain flexible loop pro-UKmutant, such as M5, or low molecular weight, two-chain pro-UK mutant,such as M5, for a time sufficient for any blood clots to be dissolved,thereby clearing the lumen of blood clots. The methods can use asolution having a concentration of mutant UK of 0.05-0.2 mg. The lumencan be in a catheter, a blood pump, or in an artificial organ, such as akidney machine.

The invention also relates to recombinant DNA methods of producingnon-glycosylated, single-chain mutants (e.g., Lys³⁰⁰→His) of pro-UK. Ingeneral, the invention features methods of preparing a pro-UK mutantpolypeptide by (a) obtaining a nucleic acid molecule that encodes apro-UK mutant polypeptide; (b) inserting the nucleic acid molecule intoa pET29a expression plasmid comprising a phage T7 promoter andShine-Dalgarno sequence; (c) transforming E. coli type B strain bacteriaBL21/DE3 RIL with the expression plasmid; (d) culturing the transformedbacteria for a time and under conditions sufficient to enable thebacteria to express pro-UK mutant polypeptide; and (e) isolating thepro-UK mutant polypeptide from the transformed bacteria.

Alternatively, one can obtain the required transformed bacteria, andfollow the same culturing and isolation steps to obtain the pro-UKmutant polypeptide. In these methods, the pro-UK mutant can be a pro-UKflexible loop mutant, e.g., M5. The pro-UK mutant can benon-glycosylated and has a molecular weight of about 45,000 daltons.

In the new methods, the culturing can be a two-stage fermentation. Forexample, the first stage of fermentation can include adding to a flask acell culture diluted in sterile EC1 medium and growing the culture at34-37° C. overnight with agitation to form a seed culture, wherein thecell culture comprises a glycerol suspension of an LB culture of thetransformed bacteria and containing a sufficient amount of kanamycin,e.g., 30 μg/ml.

The second stage of fermentation can include a) adding the seed cultureto a fermentor; b) maintaining the pH in the fermentor at about 6.5 to7.5, e.g., 6.8; c) maintaining the dissolved oxygen concentration in theculture medium at 35-45%, e.g., 40%, of air saturation; d) maintainingthe temperature of fermentation at about 34-37° C.; and e) adding to thefermentor a nutrient feeding solution, comprising one or more sugars,when all glucose initially present in the fermentor at step a) isconsumed, following the equation V=Vo e^(0.18t), where V=volume offeeding solution added (ml/h), Vo= 1/100 of the starting fermentationmedium (ml), and t=time of fermentation after the start of the feedingphase (hours). The plasmid can be pET29aUKM5, as described herein.

In some embodiments, the method can further include preparing atwo-chain pro-UK mutant, e.g., two-chained M5 (tcM5), by passing thepro-UK mutant over plasmin bound to a substrate, e.g., an agarose-basedgel filtration matrix such as Sepharose®.

In other aspects, the invention includes purified pro-UK mutantpolypeptides, such as flexible loop mutants, e.g., M5 (both as describedherein), e.g., produced according to the methods described herein. Theisolated pro-UK mutant polypeptides have a purity of 95% or greater,i.e., they are in compositions in which at least 95, 96, 97, 98, or even99% of the protein in the composition is the pro-UK mutant polypeptide.The invention also features compositions including pro-UK mutants, e.g.,made according to the new methods, and an excipient, e.g., an acidicexcipient (such as acetic acid, e.g., at a pH of 5.4), as well ascompositions including an aliquot (e.g., of 20-40 mg) of a pro-UKmutant, e.g., made according to any the new methods, packaged withdirections for use in administering as a bolus or by infusion to apatient exhibiting symptoms of a stroke or a heart attack.

In another aspect, the invention also includes a purified culture of E.coli type B strain bacteria BL21/DE3 RIL, wherein bacteria in theculture contain an expression plasmid encoding a pro-urokinase flexibleloop mutant polypeptide, such as plasmid pET29aUKM5.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are schematic diagrams illustrating a “good,” woundsealing fibrin clot (1A) and an occlusive “bad” thrombus in bloodvessels (1B).

FIGS. 2A to 2F are graphs representing in vitro lysis of ¹²⁵I-labeledplasma clots (0.2 mL) in a plasma milieu (4 mL) by M5 (3.0-14.0 μg/mL)(FIGS. 2D-F) or pro-UK (0.5-3.0 μg/mL)(FIGS. 2A-C) over afibrin-specific (<25% fibrinogen degradation) dose range. The maximalrate of lysis, as reflected by the steepest slope, was about70-100%/hour for M5 and 40-50%/hour for pro-UK. FIGS. 2B and 2E showplasminogen remaining (versus concentration of M5 or pro-UK), and FIGS.2C and 2F show fibrinogen remaining (v. concentration of M5 of pro-UK),which indicate whether lysis was fibrin-specific or non-specific. Theexperiment consists of incubating both pro-UK and M5 in human plasma for5 hours (37° C.) and then measuring the remaining plasminogen andfibrinogen. Their consumption is a measure of conversion to thetwo-chain enzyme, i.e., a measure of stability.

FIG. 3 is a graph representing in vivo lysis of ¹²⁵I-labeled whole bloodclots in the dog. The radioactivity in plasma samples (mean±SD) obtainedat intervals is shown. The number of dogs in each group is shown inparentheses and the infusate (saline, pro-UK, t-PA, or M5) and infusionrate (μg/kg/min) are indicated. Pro-UK cannot be given at the infusionrate of 60 (μg/kg/min) because it will immediately convert to UK andcause bleeding and consume plasminogen, the substrate needed for lysis.This results in less lysis (“plasminogen steal” effect).

FIG. 4 is a bar graph showing bleeding time (mean±SD) in dogs (number ofdogs tested is shown in parentheses) infused with saline, t-PA, pro-UK,or M5 (60 μg/kg/min).

FIG. 5 is a bar graph of total bleeding (mean±SD) as measured by thetotal number of gauze pads used to absorb the blood from the bleedingwounds in the dogs infused with saline, t-PA, pro-UK, or M5.

FIG. 6 is a bar graph of clot lysis, bleeding time (BT), and lysiscontrol in four monkeys infused with M5 (60 μg/kg/min). The mean±SDvalues at baseline (0), 30, 45, and 60 minutes are shown. The resultsare presented as a percent of the baseline (0% or 100%) value. The twosaline infused monkeys had ˜8% clot lysis at 60 minutes (not shown).

FIG. 7 is a schematic of an expandable intravascular balloon with acoating that contains an activated two-chain pro-UK mutant (“mutantUK”), such as two-chain pro-UK mutant. M5 (“tcM5”).

FIG. 8 is a schematic of an implantable intravascular stent with acoating that slowly releases a pro-UK mutant in a controlled, sustainedrelease.

FIG. 9 is a representation of a plasmid (pET-29a) that is used in thenew methods of making pro-UK mutants such as M5.

FIG. 10 is a representation of plasmid pET-29aUKM5 that can be used tomake M5.

FIG. 11 is a representation of a method to construct the pET-29aUKM5plasmid.

FIG. 12 is a representation of an electrophoresis gel showing themolecular weight of various proteins produced by various strains of E.coli and different plasmids.

FIGS. 13A and 13B are a pair of zymograms showing the time course of theformation of inhibitor complexes of two-chain M5 (13A) and two-chainpro-UK (13B).

FIG. 14A is a graph showing percentage of clot lysis by M5 and t-PA overtime in an arterial thrombus dog model.

FIG. 14B is a graph showing flow averages of M5 and t-PA over time in anarterial thrombus dog model.

DETAILED DESCRIPTION

Certain mutant forms of pro-UK, so-called “pro-UK mutants,” onceactivated, have a catalytic efficiency that is higher than UK. In aplasma milieu in vitro or in vivo they can induce clot lysis that istwice as rapid as pro-UK (or t-PA). The rate is sufficiently rapid toalso make the pro-UK mutants more efficient, e.g., the total amount ofpro-UK mutant needed is less than what is needed when using pro-UK ort-PA. At the same time, the wound sealing, hemostatic fibrin “good”clots are spared. The bad clots are occlusive thrombi or emboli thatinclude partially degraded fibrin. The pro-UK mutants are selectivelytargeted to the plasminogen bound to these occlusive clots. On the otherhand, the good clots are comprised of intact fibrin, and the pro-UKmutants are relatively inactive against plasminogen bound to these goodclots.

Our theory to explain how the pro-UK mutants can preferentially lyse theocclusive clots and spare the wound sealing clots is based on therealization of the importance of the fact that their substrate,plasminogen (whose activation converts it to plasmin, which lyses thefibrin in the clots), takes on three different conformations. In itsfirst conformation, also known as the “closed” conformation, plasminogenis unbound and this is the form in which it is present in the blood.

UK, the activated form of native pro-UK, activates plasminogen in thisfirst conformation, and thus causes non-specific hemorrhagic diathesis,i.e., a hemophilia-like state. Because pro-UK at therapeutic doses isunstable, it is readily converted to UK, and thus causes thishemophilia-like problem, just as when UK is administered directly. Thepro-UK mutants are designed to be much more stable than native pro-UK,and are thus not converted to UK as readily, even at therapeutic doses.As a result, they do not activate plasminogen in this firstconformation. They therefore remain inactive.

Although it has been said that plasminogen has only one fibrin-bound, or“open” conformation, we have realized the importance here of the factsthat plasminogen actually has two different and distinct conformationson fibrin and that these provide the basis for distinguishing “good”from “bad” fibrin. One conformation takes place when plasminogen isbound to an internal lysine on fibrin fragment D, which is present onintact fibrin. The other is when it is bound to the carboxy-terminallysines on fibrin fragment E. This binding site is exposed only aftersome fibrin degradation, and plasminogen bound to this site is known tobe highly preferentially activated by pro-UK, and, therefore, mutantpro-UK. By contrast, t-PA preferentially activates the plasminogen onfibrin fragment D (intact, undegraded fibrin).

This difference provides an explanation for why mutant pro-UK spareshemostatic or “good” fibrin for the following reasons. When hemostaticfibrin forms to seal an injury, it acts like a bandage and causes nointerference with blood flow. It remains intact and, by definition, hasonly the plasminogen binding site of fragment D. Pro-UK mutants, becauseof their superior stability (they remain in the pro-enzyme form), havelittle activity against this plasminogen. By contrast, when anintravascular clot forms, it impedes or arrests blood flow, whichtriggers the local release of t-PA stored in the vessel wall, and somefibrin degradation occurs. This exposes the new plasminogen bindingsites on fibrin fragment E. Plasminogen bound to these sites isespecially (˜200-300 fold more) sensitive to activation by mutant pro-UK(zymogen pro-UK) compared to plasminogen bound to fibrin fragment D. Dueto the superior stability of the pro-UK mutants, they are able toexploit this important difference between “good” and “bad” fibrin clotsat therapeutic concentrations, and thereby can induce effective clotlysis without degrading a hemostatic plug, which seals an injury.

As illustrated in FIG. 1A, when plasminogen contacts and binds to intactfibrin in a blood clot, it takes on a second conformation compared toplasminogen in circulating blood. Intact fibrin is found in newlyforming, wound sealing clots (and plasminogen binds to internal lysinesof fibrin). In this conformation, plasminogen is susceptible toactivation by t-PA, but not pro-UK mutants, which we have found spareclots that contain plasminogen in this second conformation. T-PA is notnormally present in the blood stream (only bound by t-PA inhibitors, asshown in FIG. 1A), so although plasminogen binds to fibrin in newlyforming, wound sealing clots, there is no unbound t-PA to activate theplasminogen, and so it does not lyse the fibrin. However, as soon as theblood clot grows too large or dislodges and moves to a narrower vessel,and the clot occludes the vessel, t-PA is secreted from the walls of theoccluded blood vessel and starts activating the plasminogen in itssecond conformation. The activated plasminogen is converted to plasmin,which in turn starts lysing and degrading the fibrin in the clot.

As illustrated in FIG. 1B, the degraded fibrin in occlusive blood clotsinduces the plasminogen to take on yet a third conformation, because itis now binding to additional binding sites (lysine residues on carboxyterminus of fibrin that are presented only in degraded clots) on thedegraded fibrin that are not available in intact fibrin. It isplasminogen in this third conformation that the pro-UK mutantspreferentially activate, while sparing plasminogen in its second (andfirst) conformations. Thus, the pro-UK mutants preferentially cause thelysis of occlusive, bad clots, and do not generally cause the lysis ofnewly forming, wound sealing, good clots.

We have also learned that pro-UK mutants, once activated into theirtwo-chain form, are significantly more rapidly and efficiently inhibitedthan UK (the active form of pro-UK) by a plasma inhibitor calledC1-Inactivator, which has relatively little effect on UK. TheC1-Inactivator is a substrate for two-chain pro-UK mutants, and preventsthem from binding to their natural substrate, plasminogen. The moreefficiently the two-chain pro-UK is neutralized in the blood, the lessnon-specific effects there will be, and the more the enzymatic activitywill be confined to the immediate vicinity of an occlusive blood clot.These differences represent additional evidence that the catalytic siteof two-chain pro-UK mutants is not identical to that of UK.

Pro-UK Mutants

Native pro-UK is a protein having 411 amino acids, with severaldifferent domains including the so-called “flexible loop” at amino acidlocations 297-313. The pro-UK mutants useful in the new methods,devices, and compositions described herein include those that increasethe stability of pro-UK, that can be administered at therapeutic doses,and that preferentially activate plasminogen in its third conformationon degraded fibrin found in occlusive blood clots, and spares woundsealing clots that comprise mostly or entirely intact fibrin. Thus,useful pro-UK mutants include those described in U.S. Pat. No. 5,472,692(incorporated herein by reference in its entirety), such as the pro-UK“flexible loop” mutants, which are those in which one of several aminoacids in the flexible loop of the pro-UK protein (e.g., Gly²⁹⁹, Lys³⁰⁰,or Glu³⁰¹) is replaced by a neutral amino acid such as alanine (Ala) oran amino acid that can take on only a weak positive charge, such ashistidine (His). Specific examples include Gly²⁹⁹→Ala, Lys³⁰⁰→His(referred to herein as the “M5” pro-UK mutant), Lys³⁰⁰→Ala, orGlu³⁰¹→His mutants.

The pro-UK mutants are much more stable in blood than native pro-UK,because they do not induce the formation of plasmin, and thus should beat least two, three, or four times as stable in blood than nativepro-UK. The pro-UK mutants also have a lower intrinsic activity thannative pro-UK, and once activated into their two-chain form, the pro-UKmutants can, but need not, have a higher level of activity whenactivating plasminogen. For example, the specific activity of two-chainM5 is about 100,000-200,000 IU/mg, depending on conditions and methodsof measuring, which vary from the same to double the catalytic activityof native UK.

As will be described in detail below, these pro-UK mutants are shown tohave significant advantages over pro-UK and other thrombolytic agents,and these advantages allow them to be used in a variety of new methodsand devices useful for thrombolysis and treating various cardiovasculardisorders in situations where administration of thrombolytics currentlyhas been too risky or even contraindicated, e.g., after an apparentstroke or heart attack, or in the post-operative period, a time when theincidence of thromboembolism is particularly high.

Pro-UK Mutants Dissolve Occlusive Clots More Effectively Than Pro-UK andSpare Wound Sealing Clots

One of the pro-UK flexible loop mutants, M5, has been tested in vitroand in vivo in two animal species, and shown to dissolve clots twice asfast and more efficiently (i.e., the total amount of M5 needed toachieve lysis of 50% of the lung clots in a dog was half that of pro-UK)than pro-UK. The mutant was also far more stable in blood than nativepro-UK. Of significant importance was the fact that hemorrhagic sideeffects normally associated with thrombolytic agents was not seen withthis pro-UK flexible loop mutant. These findings have been published inLiu et al., Circulation Research, 90:757-763 (Apr. 19, 2002).

In the field of thrombolysis, the “Holy Grail” has been defined asthrombolysis without bleeding, i.e., without associated interferencewith hemostasis. These two seemingly contradictory effects have neverbeen achieved before with one drug. Based on the findings describedherein, it is now clear that pro-UK mutants, e.g., the pro-UK flexibleloop mutants such as M5, can achieve this goal, and due to theirexceptional catalytic efficiency against plasminogen, can dissolve clotsfaster than other known thrombolytic agents as well.

The combination of superior safety and efficacy of specific pro-UKmutants has the potential to revolutionize the use of thrombolytics forthe treatment of heart attacks, strokes, and other blood clot-relateddiseases. Furthermore, M5 and other pro-UK mutants, such as the pro-UKflexible loop mutants, are expected to be safe for human administration,because (1) they are essentially a natural human protein (99.8% similarto pro-UK, in general, only 1, 2 or 3 of 411 amino acids are changedcompared to pro-UK), (2) they are free of antigenic (immunologic)reactions, and (3) naturally occurring human pro-UK and recombinanthuman pro-UK from E. Coli have already been safely administered to about5,000 patients in Phase III clinical studies.

New Applications of Pro-UK Mutants

The pro-UK mutants can now be used in new methods as well as in newdevices for use in various thrombolytic therapies.

Methods of Lysing Blood Clots

1. Stroke

One of the new applications is the treatment of stroke. A stroke iscaused by damage that may be either ischemic, due to a blood clotobstructing flow, or hemorrhagic, due to a broken vessel. About 70% ofthe time, a stroke is due to a clot and therefore is amenable totreatment by a thrombolytic agent, provided it is given in time. Onethrombolytic, t-PA is currently approved for this use. However, it israrely used in practice because it causes a serious brain hemorrhage inat least 10% of the cases. Since this complication is much more visiblein the individual patient than the benefit from the drug, physicianshave been reluctant to use this treatment. This high risk of hemorrhagewith t-PA is probably related to the property of t-PA, that causes it totarget intact, or hemostatic fibrin.

Unfortunately, it is difficult and time consuming to fully diagnose thecause of a stroke, but an accurate diagnosis is critical for treatmentwith t-PA or other available thrombolytic agents. Administering athrombolytic agent to a stroke victim who has a blood clot may be, atleast theoretically, a proper therapy, but administering the samethrombolytic agent to a stroke victim who has a broken blood vessel inthe brain will exacerbate the problem and can kill the patient. Althoughit takes time to confirm a diagnosis, the basic symptoms of strokeexhibited by a person (such as sudden onset of one-sided paralysis) canbe readily determined by one of skill in the medical field, such as anEMT, a nurse, or a doctor, or even a layperson with minimal training.

The new discovery that pro-UK mutants such as M5 spare hemostaticfibrin, makes it possible to treat patients with a possible ischemicstroke safely and remove the stigma and potential danger associated withcurrent thrombolytic agents. Since M5 spares hemostatic fibrin, it willnot aggravate hemorrhages in the brain. Thus, it is now possible tosafely treat all strokes immediately without delaying treatment bytime-consuming diagnostic procedures (e.g., CT scan).

Currently, pro-UK has been in clinical trials for ischemic stroke usinga complicated intra-arterial route to infuse the drug directly to thelocation of the clot. This greatly complicates the treatment, and a 10%incidence of brain hemorrhage was also found. However, unlike t-PA,which must be administered within three hours after stroke onset, theuse of pro-UK was associated with benefit up to 6 hours after strokeonset. By contrast, pro-UK mutants such as the flexible loop mutants(e.g., M5) can be given intravenously, will significantly lower theincidence of hemorrhagic complications, and extend the window of timeduring which treatment is possible well beyond that possible for t-PA,the only thrombolytic approved for this indication.

The intra-arterial administration of pro-UK mutants will also provideadditional efficacy and safety in the treatment of stroke patients. Theintravenous dose of M5 is estimated to be 120-200 mg/hour (e.g., 100,125, 150, or 175 mg/hour), whereas the intra-arterial infusion rate willbe 50-100 mg/hour (e.g., 60, 70, 80, or 90/mg/hour).

2. Heart Attack

A heart attack occurs when one of the coronary arteries is blocked,e.g., by a blood clot. The timing of reperfusion after a heart attack iscritical, because the longer the heart muscle is without oxygenatedblood, the more muscle cells are damaged and die. At present, heartattack victims are typically taken to a hospital, diagnosed, and thentreated by a coronary angioplasty, which mechanically opens the blockedcoronary artery. However, valuable time (typically 90-120 minutes afterthe onset of symptoms of a heart attack) is lost transporting thepatient to a hospital, substantiating the diagnosis, and setting up thecatheterization room and assembling the personnel to perform theangioplasty. This first 1 to 2 hours after a coronary occlusion has beencalled the “Golden Hour” because it is the time during which the maximumsalvage of heart muscle and the maximum reduction in mortality ispossible. The basic symptoms of a heart attack exhibited by a person(e.g., a typical type of chest pain associated with some shortness ofbreath) can be readily determined by one of skill in the medical field,such as an EMT, a nurse, or a doctor, or even a layperson with minimaltraining.

Unfortunately, t-PA and other thrombolytic agents are not used duringthis time because they are known to be inimical to the angioplastyprocedure (they increase both the bleeding and the clottingcomplications), which is the current standard treatment.

Based on the newly discovered property of the pro-UK mutants that theyremain stable and inert in blood and also do not lyse hemostatic, woundsealing clots, these pro-UK mutants can be safely administered topotential heart attack victims immediately after the emergency medicaltechnicians (“EMTs”) arrive on the scene. No confirmation of thediagnosis would be required, and thus patients can be treated even on amere suspicion of a heart attack. Thus, the pro-UK mutants such as M5are ideally suited for use in ambulances, and can be used to fullyexploit the therapeutic potential during the “Golden Hour.” Furthermore,by the time the patient arrives at the hospital, the occlusive bloodclot may have been dissolved by the thrombolytic therapy started in theambulance, avoiding the immediate need for an invasive angioplasty.

The pro-UK mutants can also be administered preceding percutaneoustransluminal coronary angioplasty (“PTCA”) or similar invasive vascularprocedure, to improve the benefits of PTCA by lysing the fibrin clotcomponent of the lesion, while simultaneously avoiding bleedingcomplications that could arise should the blood vessels be damagedduring the procedure. It has been shown that the results of PTCA arebetter when the clot that is associated with the atherosclerotic plaqueis removed, and thus the pro-UK mutants are especially well suited tothis adjunctive therapy.

For treating a suspected heart attack, the pro-UK mutants areadministered by intravenous infusion at a rate of 120-200 mg/hour, e.g.,140, 160, or 180 mg/hour.

3. Pre- and Post-Operative Thrombolysis

In general, thrombolytic agents are strictly contraindicated prior toand for at least 2-3 weeks after surgical procedures, because theseagents cause the lysis of hemostatic fibrin that seals the surgicalwounds. At the same time, the post-operative period is a high riskperiod for venous thromboembolism (lung clots) and also for heartattack. Unfortunately, because of the bleeding complications,thrombolytic treatment with currently available agents has not beenavailable for these patients.

The new methods include the administration of pro-UK mutants just priorto and/or after surgical procedures to treat these thromboticcomplications of surgery with little fear of causing systemic bleedingor preventing the formation of beneficial hemostatic blood clots thatseal the surgical wound. For these methods, the pro-UK mutants areadministered by intravenous infusion at 120-200 mg/hour, e.g., 140, 160,or 180 mg/hour.

For example, the pro-UK mutants can be administered after operationsknown to be associated with a high risk of embolisms, such as hipreplacement surgery, and other massively invasive procedures.

4. Clearing Lumens of Catheters and Other Devices of Blood Clots

Because mutant UKs (i.e., activated, two-chain pro-UK (tcpro-UK)mutants) are now known to have such a high efficacy and rate ofdissolving blood clots compared to other known thrombolytic agents, suchas t-PA, they can be used to clear lumens within catheters, syringes,pumps, artificial kidney machines, heart-lung machines, and other bloodtransporting devices of blood clots.

Low molecular weight (LMW) mutant UK comprising essentially thecatalytic domain of the complete mutant UK molecule, e.g., tcpro-UKflexible loop mutants such as tcM5, can be used in a way similar to theway low molecular weight UK is used in the field. Its smaller size (33Kvs. 50K) improves diffusion, which has advantages under certaincircumstances. LMW mutant UK can be obtained by cleaving mutant UK atthe Lys¹³⁵ amino acid location of the molecule, e.g., with plasmin,e.g., using Sepharose®-bound plasmin. The LMW mutant UK has the samecatalytic activity as full-length mutant UK, but due to its lowermolecular weight it diffuses better, which is advantageous for thisparticular use.

Devices That Lyse Blood Clots

1. Intravascular Balloons

The provision of a coating of the two-chain activated form of a pro-UKmutant (tcpro-UK mutant) such as tcM5, on angioplasty balloons hassignificant utility. The uniquely high activity of mutant UK makes itespecially good for balloon angioplasty to dissolve the clot that iscommonly present, the removal of which improves the results ofangioplasty and reduces the risk of re-occlusion.

In other embodiments, mutant UK can be delivered directly to anangioplasty site, e.g., by being incorporated on or in a coating onangioplasty balloon catheters and other invasive vascular devices andimplants. Such coating may be both for anti-thrombogenic purposes and toattack and dissolve pre-existing clots.

Direct delivery of the rapidly reactive mutant UK to an occludedvascular site combined with an invasive vascular procedure to removeclot or reform plaque or other stenotic structures providessignificantly improved results. In particular, a combined or consecutivelocal administration of mutant UK and balloon angioplasty provides ahighly effective cooperative action.

The combination has practical advantages. While it is effective toemploy the same balloon for both drug delivery and vessel expansion,direct delivery of mutant UK to an occlusion in other ways is seen alsoto lead to useful benefit. For instance, mutant UK can be delivered by adrug-delivery guide wire during its placement, or on a placement tubeduring its introduction, as a step preceding moving the balloon to thesite. In another case, a mutant UK delivery probe may be deployed over aguide wire or through a placement tube before insertion of the ballooncatheter. In cases involving little or no delay between introduction ofa fibrinolytic agent and balloon angioplasty, a distal, non-inflatableend portion of a balloon catheter can be constructed with a deliverylayer such as described below. Such end portion is thrust against anoccluding clot as the distal end of the catheter shaft arrives andwedges into a stenotic region. The squeezing pressure of this forwardthrust can deliver the suspension of mutant UK to an occluding clot orother stenotic region, e.g., a site for stent placement. In other cases,special purpose mutant UK delivery balloons, located either distally orproximally of the angioplasty, dilatation, or stent-placement balloon onthe balloon catheter shaft may be utilized.

In one implementation (shown in FIG. 7), the angioplasty, dilatation, orstent-placement balloon 4 itself forms a part of the direct drugdelivery system. The balloon catheter can be made of any conventionalform, e.g., suited for insertion by any percutaneous or cut downprocedure. It is constructed typically to be introduced over a guidewire and/or a placement tube or catheter 3. The exterior of the balloonis provided with a carrier layer 6, such as a swellable polymer hydrogelcoating, containing a suspension of mutant UK, which is released byapplication of pressure upon the carrier layer 6 by expansion of theballoon 4 against the walls 9 of the stenotic or occluded region 1 e. Asshown in FIG. 7, the balloon 4 is expanded against plaque 5 on vesselwall 9.

In certain implementations, the carrier is compressible or sponge-like,while in others the layer may be less compressible or incompressible,with open pores that upon application of pressure on a clot or stenoticregion forces portions of the clot to enter the pores and contact themutant-UK agent.

In some embodiments, the carrier uniformly surrounds the balloon, and inother embodiments it is an attached layer or membrane.

We note that many balloons for angioplasty are formed of inextensiblematerial, such as bi-axially oriented polyester tubing. The surface ofsuch material may be metallized, as with tantalum, to form tantalumdioxide when exposed to oxidizing conditions, following which a thinlayer of nitrocellulose may be applied, capable of carrying proteinssuch as provided here. The thickness of the coating may be one or a fewthousandths of an inch in thickness, to tightly conform to the balloon.Even in such small thickness, it may be rendered porous, in the knownway of evaporation techniques of the first of two solvents from a slurryof nitrocellulose. The porosity provided in this manner enhances thecarrying capacity of the film for the suspended enzyme.

Direct administration of the mutant UK by such means as described inconjunction with the reformation of angioplasty or dilatation, has manyanticipated advantages: lysis of clot; reduction of stenosis; clearingof clot from plaque thereby lowering the risk of clot regrowth andrestenosis; and to provide an improved plaque bed for seating acontemporaneously or subsequently placed stent. All of this is foreseento occur with little deleterious effect. Blood inhibitors of mutant UKand plasmin (such as the C Inhibitor) will ensure that the fibrinolyticeffect remains local, thereby eliminating bleeding risk.

In regard to details of construction, the drug delivery device of anyform is constructed to protect the mutant UK from substantial reactionwith blood constituents during introduction, prior to balloon inflation.In the case of a drug delivery guide wire or placement tube, the drugmay be contained within a lumen of the guide wire or tube and thendelivered in a radial or axial jet in a selected or in all directions byactivation of a plunger via a flexible push wire that extends the lengthof the guide wire to the operator. Other pumping or displacement actionsdescribed in the literature may likewise be employed. In the case of anindependent drug delivery device operating over a guide wire, it too mayhouse the drug during transit, and protect it from interaction withblood. Its larger size enables more complex jetting arrangements. Thesemay be used in conjunction with expansible members or a balloon that mayspread apart the clot and plaque to enable better application of theagent, but without the expansion force associated with true angioplasty.

In the case of balloon delivery of mutant UK, carrier-pore retention andcompressible micro-chamber arrangements are presently favored to carryand protect the mutant UK during transit to the site.

In one arrangement, a thin hydrophilic layer is provided over theexterior surface of an angioplasty balloon. It is formed for instance ofnitrocellulose by the known film-form dipping of the balloon, followedby differential solvent leaching to leave an open, porous structure. Thelayer is constructed to carry the suspension of mutant UK in the depthof its pores, in a position relatively protected from blood, andsufficiently bound to the porous structure to avoid excessive lossduring insertion movements. The layer is constructed to bepressure-sensitive for release of the agent with application of modestballoon pressure. By preliminary inflation of the angioplasty balloonagainst the stenotic walls, the suspension of mutant UK may be expressedfrom the pores and applied to the clot and/or plaque. To prevent escapeof the suspension at the ends of the elongated balloon, circumferentialend bands of solid coating, not porous, are provided to block end-wiseflow. Thus, the mutant UK suspension flows outwardly to the surroundingtarget. The thickness of the porous delivery layer may be 0.5 millimeter(0.020 inch) or more or less, the selection of thickness being relatedto the size of the balloon (thicker layers more tolerable in largerballoons), the quantity of drug to be delivered, and its level ofdilution. For example 0.1 mg/ml can provide a total dose of 0.05-0.1 mg.In other examples, the porous layer is formed of porous polystyrene orother biocompatible drug carrying materials. In another case, thedelivery layer is an open cell hydrophilic foam adhered to the exteriorwall of the balloon. In all such cases a lubricious surface (e.g., ofhydrophilic polymers or polytetrafluoroethylene) may be provided to aidtransit during the sliding movements of insertion.

In another arrangement, a set of micro-chambers formed by highlyflexible plastic film, capable of being filled with the mutant UKsuspension, are secured about the exterior surface of the balloon. Forinstance, the micro chambers may be of parallel tubular form inlongitudinal or circumferential array. The chamber walls are relativelyweak, as at scored lines, adapted to rupture under selected pressure torelease the mutant-UK suspension in a well-dispersed pattern. In thisform, a lubricious coating may be thoroughly applied to the exterior ofthe balloon for ease of insertion.

In another arrangement, a highly perforated membrane is adhered at a setof closely spaced continuous parallel lines about the balloon, to formelongated flute-like micro-chambers. The membrane has a low, but mutantatmospheric break-through pressure. At atmospheric pressure, and duringtransit through the vascular system, the walls inwardly contain andprotect the suspension by surface tension at the pores. However, underpressurized expansion of the balloon against the vascular wall, thepores of the membrane enlarge. Break-through pressure is exceeded,enabling radial flow of the suspension through the pores of the walls asthe chambers are compressed under pressure. Such a perforated film maybe provided as a thin-walled exterior electrometric balloon, fittedabout a conventional, preformed non-elastic polymer balloon, e.g., madeof polyethylene terephthalate (PET). The micropores in the membrane maybe produced by a perforating laser or electron beam, or, duringformation of the membrane by inclusion of porsigens in a fluid polymercomposition of which the membrane is formed. By the sets of flute-formor tubular chambers, or other dividers, uniform dispersion of the mutantUK or M5 suspension around the balloon is provided.

It is feasible to pre-load catheters with the drug in lyophilized form,and store the assembly under suitable drug-storage conditions.

In such cases only sterilized saline or similar fluid carrier need beintroduced to create the deliverable suspension prior to use. In othercases, here and with other interventional devices or implants,lyophilized mutant-UK is carried on the surface, and blood in thevessel, e.g., at the site, is relied upon for delivery of thefibrinolytic agent.

However, in presently preferred cases, the mutant UK is packagedseparately as a drug in unit dose and applied to the delivery system bythe attending scrub nurse or catheter technician. It is stored as alyophilized preparation not requiring refrigeration, and prepared insuspension, e.g., by the pharmacy. In this case, the attendant appliesthe drug suspension to the hydrophilic sponge layer or other carrier byan applicator syringe squeegee, which may be an integrated part of thedrug package. By rotating a balloon catheter by hand, during applicatorstrokes, uniform coverage of the surface of a balloon can be obtained.In another case, a close-fitting mold-like container or elastic sleeveis provided into which the balloon end of the catheter is placed and thesuspension is poured or applied by a needle syringe about the insertedballoon. The balloon is slightly inflated, and the balloon pushed andpulled repeatedly within the container or sleeve to uniformly distributethe mutant UK suspension over the delivery layer.

In the fluted or tubular chamber versions previously described, theattendant inserts the needle of a syringe to fill each chamber, or thechambers are interconnected so that only one needle insertion isrequired. An advantage in forming the chambers of porous membrane isthat displaced air readily escapes directly through the walls. If thechambers are formed by impermeable film, a permeable air release sectionis provided at the end of the volume remote from its fill point. In allsuch cases, care is taken to remove all air.

In employing the various arrangements described, or using other knowndrug delivery systems on balloons, etc., mutant UK, e.g., tcM5, isdelivered directly to the clot and plaque before major angioplasticdeformation. For application simultaneous with angioplasty, the timingand duration of drug release relative to increasing balloon pressure canbe arranged to enable continued delivery of the mutant UK suspension upto the highest pressure phase of balloon and plaque expansion.

In other arrangements, a drug-delivery balloon, such as one of thosedescribed, but not constructed for angioplasty, is mounted on a cathetershaft distal of an angioplasty or dilatation balloon. The drug deliveryballoon is first positioned at the operative site and inflatedsufficiently to apply the mutant. UK. The drug delivery balloon isdeflated, the catheter advanced to position the angioplasty balloon, andangioplasty is completed using an unmodified angioplasty or dilatationballoon. In other arrangements, the drug delivery balloon is located ona shaft proximally of an angioplasty or dilatation balloon. The latteris first advanced past the operative site to position the drug-deliveryballoon, mutant UK is delivered, the drug delivery balloon deflated, andthe angioplasty catheter is withdrawn to position an unmodifiedangioplasty or dilatation balloon at the operative site, and the balloonangioplasty expansion procedure completed. In another arrangement, thedrug delivery system is either provided on the balloon, or distally orproximally as a separate balloon, in one of the manners describedherein, while a stent is carried in another location on the samecatheter, in ways that are known, so that combined mutant UK directdelivery, angioplasty or dilatation, and stent placement can occur withonly one catheter insertion. In some cases, the sequence may beperformed in any order, or drug delivery may be performed repeatedly inany order.

In one advantageous case, mutant UK is delivered before orsimultaneously with balloon angioplasty, stent placement is performed,e.g., by a self-expanding stent such as formed of mutant-elastic metal,and following placement of the stent, the agents either mutant UK (e.g.,tcM5), for immediate action, non-activated, single-chain M5 foradherence and prolonged action, or a mixture, are applied to theinterior of the stent after placement. For this purpose, advantageously,the stent is provided with a receptive, or in other cases an enzymereceptive and retentive surface, so that post placement application ofM5 to the stent results in retention of M5 at the stent for combatingclot formation or restenosis, or for gradual, eluted delivery to thevascular system.

Other delivery interventional and implant placement techniques maylikewise be employed in combination with mutant UK (e.g., tcM5) andnon-activated M5. Reference is made for instance to U.S. Pat. Nos.6,409,716 and 6,364,893.

2. Stents and Other Implants

The stability of the pro-UK flexible loop mutants, such as M5, make themespecially attractive for use on the surface of stents, since they arestable and inert in blood, thus allowing them to be slowly released in asustained fashion from coatings on stents after implantation. Becausethe pro-UK mutants are inert until they contact a blood clot, they willhave no undesirable side effects if no clots are present, but will beimmediately and locally activated, and thus effective in the vicinity ofthe stent if a blood clot should begin to form on or near the stent.This will prevent re-occlusion and diminish the need of using costlyadjunctive therapy with anti-thrombotic agents, such as the IIb and/orIIIa inhibitors, which also carry a risk of hemorrhage andthrombocytopenia.

FIG. 8 illustrates a stent 10 and a placement balloon catheter 12 afterthe balloon catheter has been deflated and is being pulled out of thestent. The stent is compressing the occlusion 15 on blood vessel wall 9.Each wire or plastic fiber of the stent can be coated with a carrierlayer that contains a pro-UK mutant, or the stent can be “lined” with acoating layer that is transported by an expandable balloon catheter andthen left behind with the stent when the balloon is withdrawn, e.g., asdescribed in U.S. Pat. No. 6,364,893.

Following angioplasty or dilatation, placement of a stent to protectagainst re-occlusion should be facilitated by the prior orcontemporaneous application of mutant UK or M5 as described. Adrug-eluting stent can be used, carrying a durable dosage of M5, thezymogenic, single-chain precursor of mutant UK. It is anticipated thatthis will inhibit clot formation during the early, critical phase whenrisk of re-clotting is the greatest. Release of M5 will prevent thelocal deposition of fibrin. In the circulation, M5 will be harmlessbecause it is inert in the absence of fibrin.

The pro-UK mutants can be provided in a varying or homogenousdistribution across the thickness of the biocompatible coating or layerto act, or be released, over time in the vascular system or in passagescarrying blood. Such coatings can be prepared and applied to variousdevices using standard techniques. These coatings provide a fresh,unactivated pro-UK mutant such as M5 to act upon blood clots in theimmediate vicinity of the device. Known inhibitors of activated;two-chain pro-UK mutants, such as C1 Inactivator, strongly inhibit tcM5,and thus provide another mechanism to confine the activated tcpro-UKmutants to the vicinity of the devices (because they inhibit tcM5elsewhere in the blood).

Substances that can serve as the coating materials for devices will varydepending on the character or function of the device on which thecoating is placed and the site of the clot, or regions susceptible toclotting. For instance, the coating material will be different forapplication on an elastic balloon (e.g., may be elastic or highlyyieldable) versus that for application to a relatively stiff cathetershaft (need not have great elongation). For example, hydrogel coatingmaterials that can be used with pro-UK mutants are described in U.S.Pat. Nos. 6,409,716 and 6,364,893.

The stent may take any of a number of conventional forms. In one case,the stent is a so-called “Palmaz” stent, available from Johnson andJohnson. It is formed as a small, expandable cylinder of biocompatiblemetal, and has suitably positioned slits. It is sized to be inserted ona placement catheter and introduced percutaneously, and via the vascularsystem, to the position for placement following balloon angioplasty. Theslits are sized and configured to enable the cylinder, when internallystretched by inflation of the placement balloon, to form an “expanded”metal apertured cylinder. In its expanded state, it is constructed toprovide support to the vessel wall and resist collapse.

In another case, the stent is a network of super-elastic metal wirematerial, forming an open structure, of size suitable to be insertedthrough the vascular system in its reduced size state, and adapted,subsequently, e.g., when heated to body temperature, to expand to itsenlarged, vessel wall-supporting size.

In such cases, the stent structure is provided, according to theinvention, with a section whose surface, or the entire surface of thestent, is covered with a compliant, ablatable, biocompatible, andbio-erodible coating, adapted to remain intact after expansion of theballoon. Through the thickness of this coating is distributed the mutantpro-UK. Over time, as the bio-erodible, coating sluffs away, freshmutant pro-UK is exposed and made available to act upon any adjacentclot. In some cases, the coating may be a lubricious hydrophiliccoating, such as described in the referenced patents.

Thus, the stents are of a form familiar to the interventionalcardiologist, and constructed to be inserted in a conventional manner.The stent may be pre-prepared with the coating loaded with pro-UKmutant, or mutant UK (or a mixture), and maintained under suitablerefrigeration. In other cases a pre-cursor coating is applied, and justprior to use, an aqueous suspension of lyophilized mutant is applied asby needle injection at selected locations. In other cases, a curablecoating-forming solution is prepared, through which the pro-UK mutant ormutant UK is distributed. Preparatory to the procedure, the coating isapplied by an attending technician to the stent surface and allowed tocure, as by exposure to curing agent, air, moisture, or ultravioletlight, depending upon the composition, following which the stent may beintroduced.

In addition, given their newly discovered properties, the pro-UKflexible loop mutants can be used in anti-thrombogenic coatings on otherdevices that are constructed to be used in the vascular system. Forexample, the pro-UK mutants can be included in hydrogels and otherbiocompatible coating materials that can be sprayed, painted, dipped, orotherwise applied to these devices. Specific examples of devicessuitable for use with such coating include: vena cava filters, by-passshunts, guidewires, catheters, grafts, sutures, valves, artificialhearts, and implanted drug delivery devices that administer controlleddosages by active pump action or passively by biodegradation. A shunt isa plastic tube, usually connecting a vein with an artery, e.g., toprovide access for kidney dialysis. Since plastic is “foreign” to thebody, it stimulates the blood to clot. Thus, a coating of the pro-UKmutants, e.g., M5, can dissolve these types of clots as they form. Toclear a shunt of existing blood clots, one would use mutant UK, e.g.,tcM5.

Implants may be advantageously located where infusion devices have beenknown to be effective, e.g., subcutaneous portal pump devices connectedby catheter to an artery or vein. In many cases, the regions most atrisk for forming clots are at the transition from natural tissue tosynthetic. The tissue in such regions, until stabilized during healing,is under stress. It is believed, t-PA will emerge from such tissue andinitiate the degradation of the clot, by which the fibrinolytic actionof the mutants provided is given opportunity to act. Moreover, we notethat this application takes advantage of the property of the pro-UKmutants that they are inert and inactive except in the presence of afibrin clot.

Summary of New Applications

The newly discovered properties of safety and efficacy allow the pro-UKmutants to be used in a variety of new methods and devices, and many ofthese uses are contraindicated for known thrombolytic agents. Table 1below summarizes and exemplifies some of these new uses. TABLE 1Clinical Implementations of Pro-UK Mutants and Mutant UK Compared toKnown Thrombolytic Agents Current Thrombolytic Agents (SK, t-PA,Reteplase, TNK-t- M5 Clinical Condition PA) (projected) 1. Heart AttackGeneral Used in ˜50% eligible 75-90% Pts. & declining Elderly Notadvised Advised 2. Pre-PTCA Contraindicated Indicated With-PTCA Not usedIndicated for existing clot and antithrombogenisity Post-PTCA Not usedIndicated due to Antiplatelet effect 3. Stroke Limited use within 3 hrsWell suited for use onset within 6 hour onset 4. Pulmonary Used only formajor Most emboli embolism emboli (<10%) 5. Deep vein Rarely used Manypatients thrombosis 6. Peripheral Intra-arterial t-PA or UKIntra-arterial or arterial used Intravenous occlusions 7. With Angio-Not used Indicated plasty of peripheral vesselsMethods of Making Pro-UK Mutants

Because of these unique characteristics and new methods of use, there isa need to produce single-chain pro-UK mutants, e.g., flexible loopmutants such as M5, in high quantities, at a high level of puritysufficient for administration to human patients, and with the properprotein refolding.

As noted above, pro-UK mutants are proteins that are identical to nativepro-UK, but for one or more mutations, such as a single point mutation,e.g., at one or more of the amino acids in the flexible loop (amino acidlocations 297-313), e.g., at Lys³⁰⁰; Gly²⁹⁹, or Glu³⁰¹ with a simple,neutral amino acid such as alanine (Ala), glycine (Gly), and valine(Val), or a weakly positively charged amino acid such as histidine(His). Examples of flexible loop mutants include Lys³⁰⁰→His, Lys³⁰⁰→Ala,Gly²⁹⁹→His, and Glu³⁰¹→Ala.

The pro-UK mutants must have the following characteristics: they mustincrease the stability of native pro-UK in plasma or blood by at least 3times; they must enable administration in therapeutically effectivedosages; and they must preferentially activate plasminogen in a thirdconformation found on degrading fibrin clots (occlusive clots) and spareplasminogen in its second (and first conformations) found on woundsealing clots (and floating freely in the blood). The pro-UK mutants canbe flexible loop mutants, such as M5. They can all be made using variousstandard techniques that can be scaled up for commercial applications.

Site-Directed Mutagenesis

The pro-UK mutants can be made using site-directed mutagenesis, such asoligonucleotide-directed mutagenesis, which allows the specificalteration of an existing DNA sequence, e.g., native pro-UK. The geneencoding native pro-UK is well-characterized and is available, e.g.,from Primm (Milano, Italy) or from the ATCC at Accession Nos. DNA 57329or Bact/phage 57328. The sequence is also available from the NIHcomputer database Protein Identity Resource under the name UKHU. See,also, U.S. Pat. No. 5,472,692.

In general, oligonucleotide-directed mutagenesis is accomplished bysynthesizing an oligonucleotide primer whose sequence contains themutation of interest, hybridizing the primer to a template containingthe native sequence, and extending it, e.g., with T4 DNA polymerase. Theresulting product is a heteroduplex molecule containing a mismatch dueto the mutation in the oligonucleotide. The mutation is “fixed” uponrepair of the mismatch in, e.g., E. coli cells. The details of thismethod are described, e.g., in Ausubel et al. (eds.), Current Protocolsin Molecular Biology, Chapter 8.1 (Greene Publishing Associates 1989,Supp. 13). The details of this method are generally routine, and aredescribed in U.S. Pat. No. 5,472,692.

Several variations of in vitro mutagenesis by primer extension thatyield mutants with high efficiency have been developed, as described inSmith, Ann. Rev. Genet., 19:423-463 (1986), and various methods can beused to prepare the pro-UK mutants. One example of a simplesite-directed mutagenesis protocol applied to a uracil-containingtemplate, which allows rapid and efficient recovery of mutant DNAs, isdescribed in Kunkel, Proc. Natl. Acad. Sci. U.S.A., 82:488-492 (1985),and Kunkel et al., Meth. Enzymol., 154:367-382 (1987).

Once the pro-UK DNA with the desired mutation is obtained, it must becloned into a suitable expression vector. This vector must then beintroduced into a cell line, e.g., bacterial, mammalian, or yeast, toexpress the pro-UK mutant, which is harvested from the culture medium,or from the yeast cells, and then purified. These techniques are wellknown to those of ordinary skill in the field of molecular biology andare described in detail, e.g., in Ausubel et al. (eds.), CurrentProtocols in Molecular Biology, Chapters 9 and 16, supra; and Sambrook,Fritsch, and Maniatis, Molecular Cloning (2d ed.), Chapter 16 (ColdSpring Harbor Laboratory Press, 1989). The advantage of using mammaliancells, such as Chinese Hamster Ovary (CHO) cells, to express the pro-UKmutant proteins is that they will have a mammalian glycosylation patternthat bacterial and other cells cannot provide. In vitro glycoproteinremodeling can also be used to achieve proper glycosylation when usingnon-mammalian (or low yield mammalian) cell systems.

There are several ways in which the mutant pro-UK gene can be introducedinto a mammalian cell line. As noted, one method involves thetransfection of a vector into Chinese hamster ovary (“CHO”) cells. Inthis procedure, the mutant pro-UK gene is co-transfected with aselectable marker, becomes stably integrated into host cell chromosomes,and is subsequently amplified. The CHO cell system is useful because itallows the production of large amounts of mutant pro-UK for long periodsof time.

Another expression method involves the transfection of the vectorincluding the mutant DNA into a PET-19B E. coli expression system(Novagen, Madison, Wis.). For example, after site-directed mutagenesis,the DNA encoding a Lys³⁰⁰→His mutant can be sequenced to ensure that themutation had occurred, and then ligated into the NdeI/XhaI cite ofPET-19B, and transformed into E. coli. The transformed E. coli are thencultured and induced to express the pro-UK mutant by addition of theinducer IPTG at log phase.

Once a vector has been introduced into a mammalian cell line, it alsomay be desirable to increase expression of the desired protein, e.g.,the mutant pro-UK, by selecting for increased copy numbers of thetransfected DNA within the host chromosome. Co-amplifying transfectedDNA results in a 100- to 1000-fold increase in the expression in theprotein encoded by the transfected DNA. There are more than 20selectable and amplifiable genes that have been described in theliterature, but the most experience and success has been withmethotrexate selection and amplification of transfected dihydrofolatereductase genes. For example, dihydrofolate reductase-deficient CHOcells may be used to obtain high level of expression of mutant pro-UKgenes through co-amplification by selection for methotrexate resistance.

After the mutant pro-UK is expressed, e.g., by a mammalian or bacterialcell line, it must be extracted from the culture medium and purified. Inthe case of yeast cell culture, the yeast cells must first be disrupted,e.g., by mechanical disruption with glass beads to produce a cellextract that contains the mutant pro-UK. Purification of active mutantpro-UK from culture medium or cell extracts generally involves the stepsof 1) liquid/liquid phase extraction or an initial filtration step, 2)hydrophobic affinity chromatography, 3) antibody affinitychromatography, and 4) gel filtration chromatography. These steps arewell known to those of ordinary skill in the field of molecular biology,and are described in detail in Current Protocols in Molecular Biology,Chapter 10.

Commercial Scale Production

Although general known methods can be used to make the pro-UK mutants,exceptionally high yields and purity can be achieved, e.g., at acommercial scale, using the new methods of production described herein.

The goal in these new methods is to obtain high expression and highyields of properly folded polypeptides from fermentation andpurification procedures. These goals are achieved by careful selectionof a combination of specific process parameters such as the type ofbacterial strain, the particular expression plasmid, the specificpromoter sequences, and the type of cell fermentation and proteinpurification techniques. By properly selecting these variables,recombinant bacteria are able to synthesize large amounts of the pro-UKmutant, e.g., flexible loop mutant, polypeptides, and it is possible toobtain the pro-UK mutants at high levels of purity. By employing theseselected procedures, the properly folded pro-UK mutants, such as M5, canbe produced at greater than 96, 97, 98, or even 99% of purity (i.e.,they are in compositions in which at least 96% or greater of the proteinin the composition is the single-chain pro-UK mutant polypeptide).

To isolate the desired recombinant E. coli strains, it is necessary togo through a number of steps including: (1) mutagenizing the humanpro-UK cDNA gene to isolate the desired M5 or other mutant gene; (2)inserting the mutated gene in an appropriate expression plasmid; (3)transforming a selected strain of E. coli with the engineered plasmid;(4) fermenting the transformed cells under appropriate conditions; and(5) isolating the pro-UK mutant protein. Each of these steps will bedescribed in detail.

1) Mutagenesis

The human pro-UK cDNA gene can be treated as described below to isolatethe desired pro-UK mutant encoding gene. The general methods describedin U.S. Pat. Nos. 5,866,358 and 5,472,692 can be applied to prepare thenucleic acid molecule encoding the particular desired pro-UK mutantpolypeptide.

For example, the pro-UK mutants can be made using site-directedmutagenesis, such as oligonucleotide-directed mutagenesis, which allowsthe specific alteration of the existing native pro-UK nucleic acidsequence. The gene encoding native pro-UK is well characterized and isavailable, e.g., from Primm (Milano, Italy) or from the ATCC atAccession Nos. DNA 57329 or Bact/phage 57328. The sequence is alsoavailable from the NIH computer database Protein Identity Resource underthe name UKHU. Production of a gene encoding M5 is described in U.S.Pat. No. 5,472,692.

In general, oligonucleotide-directed mutagenesis is accomplished bysynthesizing an oligonucleotide primer whose sequence contains themutation of interest, hybridizing the primer to a template containingthe native sequence, and extending it, e.g., with T4 DNA polymerase. Theresulting product is a heteroduplex molecule containing a mismatch dueto the mutation in the oligonucleotide. The mutation is “fixed” uponrepair of the mismatch in, e.g., E. coli cells. The details of thismethod are described, e.g., in Ausubel et al. (eds.), Current Protocolsin Molecular Biology, Chapter 8.1 (Greene Publishing Associates 1989,Supp. 13). The details of this method are routine, and are described inU.S. Pat. No. 5,472,692.

Several variations of in vitro mutagenesis by primer extension thatyield mutants with high efficiency have been developed, as described inSmith, Ann. Rev. Genet., 19:423-463 (1986), and various methods can beused to prepare the nucleic acid molecules encoding the pro-UK flexibleloop mutants. One example of a simple site-directed mutagenesis protocolapplied to a uracil-containing template, which allows rapid andefficient recovery of mutant DNAs, is described in Kunkel, Proc. Natl.Acad. Sci. U.S.A., 82:488-492 (1985), and Kunkel et al., Meth. Enzymol.,154:367-382 (1987).

2) Insertion of Mutant Gene into Expression Vector

Once the pro-UK DNA with the desired mutation is obtained, it must becloned into a suitable expression vector. In particular, plasmid pET29a(kanamycin-resistant), which is shown in FIG. 9 can be used (availableform Novagen). For example, pET29aUKM5 (which encodes the M5 pro-UKmutant)(FIG. 10) can be used as the expression vector to produce thespecific pro-UK flexible loop mutant M5. In this plasmid, the geneencoding M5 is inserted into the Nde I-Sac I site (see FIG. 10) on theplasmid using standard techniques. The pET-29a plasmid includes specificPhage T7 promoter and Shine-Dalgarno sequences (see, e.g., (Moffatt, B.A. and Studier, F. W. (1986). J. Mol. Biol. 189, 113-130, Rosenberg, A.H., Lade, B. N., Chui, D., Lin, S., Dunn, J. J. and Studier, F. W.(1987) Gene 56, 125-135, Studier, F. W., Rosemberg, A. H., Dunn, J. J.and Dubendorff, J. W. (1990) Meth. Ezymol. 185, 60-89)). The promoter isresponsible for the synthesis of messenger RNA while the Shine-Dalgarnosequence should guarantee an efficient translation of the mRNA in thepolypeptide chain.

Although this particular plasmid and sequences are known, and thetechniques to combine these sequences and plasmids are also well knownto those of ordinary skill in the field of molecular biology, thespecific combination of these parameters has not been described prior tothe present disclosure. The general techniques are described in detail,e.g., in Ausubel et al. (eds.), Current Protocols in Molecular Biology,Chapters 9 and 16, supra; and Sambrook, Fritsch, and Maniatis, MolecularCloning (2d ed.), Chapter 16 (Cold Spring Harbor Laboratory Press,1989).

3) Transformation of the Plasmid into a Host Cell

Next, an E. coli type B strain, BL21/DE3 RIL, is used for the expressionand production of the pro-UK mutant. For example, insertion of plasmidpET29aUKM5 into E. coli type B strain BL21/DE3 μL (available, e.g., fromSTRATAGENE®, USA) induces very high levels of expression of the M5polypeptide. Interestingly, insertion of the same plasmid into otherstrains of E. coli (type K-12, type C, or type W, and even other type Bstrains) does not provide as high a yield of M5.

For example, competent cells of strain BL21/DE3 RIL can be preparedusing a calcium chloride procedure of Mandel and Higa (Mol. Biol.,53:154, 1970). A small aliquot, e.g., 200 μl, of a preparation of thesecells, e.g., at 1×10⁹ cells per milliliter, can be transformed withplasmid DNA (approximate concentration from 2 to 10, e.g., 5 μg/ml).Transformants containing the kanamycin resistant plasmids are selectedon plates of L-agar containing 30 μg/ml kanamycin.

One or more small colonies are streaked, e.g., with wooden toothpicks,onto L-agar containing the same antibiotic. After incubation at about34-37° C., e.g., for a time sufficient to establish colonies, e.g.,about 8, 10, 12, 15, or more hours, portions of the streaks can betested for pro-UK mutant production by inoculation into LB medium(containing kanamycin at a concentration of 30 μg/ml) and incubatedovernight (e.g., 8, 10, 12, or 15 or more hours), again at about 34-37°C. The following day, the cultures can be diluted, e.g., 1:100, inmedium, such as M9 medium, containing the same concentration ofkanamycin, and incubated, e.g., for 4, 6, or 8 hours at 34-37° C.

Total cell proteins from aliquots of culture medium (O.D.₅₅₀=1 to 1.5)can be analyzed by sodium dodecylsulfate polyacrylamide gelelectrophoresis (SDS—PAGE) as described in Laemmli, Nature, 227:680,1970. A major protein band having a molecular weight corresponding tothat of non-glycosylated M5 (45,000 daltons) should be observed for thesamples.

FIG. 12 shows typical SDS-PAGE results. Lane 1 includes molecular weightstandards. Lane 2 contains BL21(DE3)RIL[pET29a] supernatant of cells atan OD of 1.5. Lane 3 contains BL21(DE3)RIL[M5-PUK] supernatant of cellsat an OD of 1.5. Lane 4 contains BL21(DE3) [M5-PUK] supernatant of cellsat an OD of 1.5. Lane 5 contains BL21(DE3)RIL[pET29a] inclusion bodiesof cells at an OD of 1.5. Lane 6 contains BL21(DE3)RIL[M5-PUK] inclusionbodies of cells at an OD of 1.5. Lane 7 contains BL21(DE3) [M5-PUK]inclusion bodies of cells at an OD of 1.5. As explained in furtherdetail in Example 13, below, these results indicate that M5 is aninsoluble protein, and only the combination of the pET-29aUKM5 plasmidwith BL21(DE3)RIL produced large quantities of M5 (Lane 6).

Using the procedure described above, several additional E. coli hoststrains were screened with the objective to isolate a transformantstrain able to produce M5 at high levels. Plasmid pET29aUKM5 wastransformed into the following strains: BL21/DE3, BL21/DE3 pLys,JM109/DE3, and HB 101/DE3. None of these transformed strains was able toyield high quantities of the M5 polypeptide as seen with the host strainBL21/DE3 RIL, indicating that indeed the combination of the specificexpression plasmid with strain BL21/DE3 RIL is an important combinationto obtain high quantities of M5. The details of these tests aredescribed in Example 14, below.

We note that the use of B strains according to the present inventionyields cell extracts with low proteolytic activity, i.e., lowcontamination with UK.

4) Fermentation of the Host Cells

The transformed bacterial cells must then be cultured at high biomass inappropriate fermentors. The protocol developed and used for theproduction of pro-UK flexible loop mutants is based on the following twostages of fermentation.

A. First Fermentation Stage (Seed Culture)

The first fermentation phase is carried out in flasks to obtain a seedculture large enough to inoculate the production stage (secondfermentation stage). One vial of “working cell bank” (e.g., 0.1 ml-1.0ml) is diluted in an amount (e.g., 50 to 500 ml, e.g., 100 ml) ofsterile medium (e.g., EC-1 medium, details of which are provided inTable 4 in Example 15, below) and growth at about 37° C. overnight withthe agitation.

The working cell bank is made of a glycerol suspension of an overnightculture (e.g., LB medium) of the pro-UK flexible loop mutant producingstrain (e.g., BL21/DE3 RIL carrying plasmid pET29aUKM5 that encodes M5),and containing an antibiotic to select for bacteria carrying a resistantplasmid (e.g., kanamycin at 30 μg/ml, chloramphenicol at 30 μg/ml, and0.1% w/v D-Glucose).

B. Second Fermentation Stage

The second stage includes the following steps:

1) the seed culture, prepared in a flask, is added to a fermentor (e.g.,a 1:100 dilution in EC-1 medium; e.g., 20 ml into 2.0 liters or 100 mlinto 10 liters);

2) the pH in the fermentor is kept at about 6.8 to 7.2, e.g., 7.0, forexample, by using a solution of 28% (v/v) ammonia water;

3) dissolved oxygen is maintained at about 35 to 45%, e.g., about 38,40, or 42%, of air saturation by increasing the agitation speed and bychanging the percentage of pure oxygen;

4) the temperature of fermentation is kept at about 34-37° C.; and

5) a nutrient feeding solution that contains one or more sugars (e.g.,glucose) and other nutrients (for a specific example, see Table 5 inExample 15, below) is added exponentially when all the glucose initiallypresent is consumed (usually after 8 hours), following the equation V=Voe^(0.18t), where V=volume of feeding solution added (ml/h), Vo= 1/100 ofthe starting fermentation medium (ml), and t=time of fermentation afterthe start of the feeding phase (hours).

In this method, gene expression is induced by adding IPTG, e.g., at afinal concentration of 1.2 mM, when the fermentation reaches a celldensity of about 90 OD₆₀₀.

The post-induction fermentation is generally prolonged for 6 hours toallow the cells to produce the pro-UK mutant. Samples of 0.5 ml areremoved from the fermentor every 2 hours for analysis.

5) Isolating and Purifying Pro-UK Mutants

After the mutant pro-UK is expressed by a bacterial cell line, it mustbe extracted from the cells and purified. Purification of active mutantpro-UK from culture medium or cell extracts generally involves the stepsof: 1) pellet recovery, 2) protein refolding, 3) concentration byultrafiltration, 4) cation exchange chromatography, 5) anion exchangechromatography, 6) hydroxyapatite chromatography, 7) gel filtrationchromatography, 8) buffer exchange, and 9) freeze drying. These stepsare described in further detail as follows.

1. Pellet recovery

In specific embodiments, the broth from a 2-liter fermentation iscollected at about 4° C. at 9950×g for 15 minutes using, e.g., a BeckmanJ2-MI centrifuge. Other methods can be used to create a pellet. Thecellular pellet is resuspended at 4° C. in 1.1 liters of a buffercomprising 0.025 M monobasic sodium phosphate, 0.125 M sodium chloride,pH 7.5 containing 0.1% Triton x 100. This slurry is passed through aFrench-Pressure-Cell 20K (Aminco) at 1000 psi. The temperature iscontrolled during this operation to about 5-10° C. After each passagethrough the French pressure cell, the cell suspension is sonicated at 15mV for about 1 minute, e.g., using a Microson™ Ultrasonic cell disrupterXL (Misonix).

Six passes under these conditions are made to achieve greater than 90%cell breakage (controlled by microscopic observation). A further passagemay be made if necessary. Other methods can be used to achieve 90% cellbreakage.

The resulting cell lysate is centrifuged at 4° C. at 9950×g for 15minutes using, for example, a Beckman J2-MI centrifuge. The solid isresuspended in 1 liter of buffer: 0.025 M monobasic sodium phosphate,0.125 M sodium chloride, pH 7.5 containing 0.1% Triton x 100. The slurryis passed again twice through the French press under the conditionsdescribed above, and centrifuged at about 4° C. at 9950×g for 15 minutesusing the centrifuge.

The slurry resulting from this process is the starting material for theactivation and purification process and is divided into suitablealiquots. Other methods can be used to obtain a similar cell lysateslurry.

2. Refolding

In specific embodiments, a quantity of the material coming from theslurry obtained, as described above, corresponding to about 6 g ofprotein (judged by Lowry or Biuret protein assay) is dissolved in a 1.2liters of 6 M guanidine HCl, 0.01 M TRIS, pH 8.5, L-cysteine 0.1 M, forat least 10 hours at 4-6° C. The solution is centrifuged at 4° C. at9950×g for 15 minutes using a centrifuge, and then diluted with 30liters of a buffer comprising 2.5 M Urea, 0.01 M TRIS, 0.005 M EDTA (pH8). The solution is stirred gently at 14-16° C. for at least 18 hours.During this period the active product is formed from the previouslyinactive protein. Other standard methods of protein refolding can beused.

3. Concentration

The solution obtained from the refolding process (2) is pre-filtered,e.g., using an Opticap® 10″ cartridge (Millipore), and thenconcentrated, e.g., about 25 times from its initial volume, using anultrafiltration system (e.g., a Millipore ProFlux® M12 with Pellicon® 2cartridges, 10,000 M.W.t. cut off). The concentrated material can thenbe diafiltrated against two volumes of a buffer consisting of 2.5 MUrea, 0.01 M TRIS, 0.005 M EDTA, and pH 7.6.

4. Cation Exchange Chromatography

The solution obtained from the concentration step (3) is centrifuged at4° C. at 9950×g for 15 minutes using a Beckman J2-MI centrifuge, andthen applied to a cation exchange column, e.g., a HiPrep™ 16/10 SP FF(Amersham Biosciences), previously equilibrated with the same dilutionbuffer. After loading, the column is washed with two column volumes ofthe equilibrating buffer followed by four column volumes of 0.01 M TRIS(ph 7.6) buffer. The column is then eluted in fractions with 0.01 MTRIS, 0.5 M sodium chloride, and pH 7.6 buffer

5. Anion Exchange Chromatography

The pool fractions from cation exchange chromatography are loaded ontoan anion exchange column, e.g., a HiPrep 16/10 Q FF (AmershamBiosciences), previously equilibrated with 0.01 M TRIS, 0.5 M sodiumchloride, and pH 7.6 buffer. The column flow-through containing theproduct is collected. After loading, the column is washed using 2-3column volumes of the equilibrating buffer.

The combined load flow-through and column wash solutions form the inputfor the next chromatographic column.

6. Hydroxyapatite chromatography

The material obtained from the anion exchange column is applied to acolumn of hydroxyapatite, such as a High Resolution (Calbiochem) column,previously equilibrated in 0.01 M TRIS, 0.5 M sodium chloride, and pH7.6 buffer. After loading, the column is washed with 2 column volumes ofthe equilibrating buffer, followed by 1 column volume of 1 mM sodiumphosphate, 0.5 M sodium chloride, pH 7.6 buffer.

The column is eluted in fractions with 20 mM sodium phosphate, 0.5 Msodium chloride, and pH 7.6 buffer. A pool of these fractions is made onthe basis of the quantity and purity judged by the specific activity.Depending on the pool volume, the resulting material may be concentratedby ultrafiltration (e.g., using a Pellicon 2 cartridge, 10,000 M.W.t.cut off) to a suitable volume for the following step.

7. Gel Filtration Chromatography

The material resulting from the hydroxyapatite chromatography is loadeddirectly (or after concentration) on to a column of HiLoad 26/60Superdex® 75 (Amersham Biosciences), previously equilibrated in 0.01 MTRIS, 0.5 M sodium chloride, 0.005 M EDTA, pH 8. The column is eluted infractions using the equilibration buffer.

Pool fractions are selected on the basis of specific activity, SDS PAGE(reduced and non reduced) and HPLC purity.

8. Buffer Exchange

The resulting material, pooled from gel filtration chromatography, isapplied to column of HiPrep 26/10 desalting (Amersham Biosciences),previously equilibrated in 0.05 M ammonium bicarbonate buffer. Thecolumn is eluted in fractions with this buffer. A pool of fractions isprepared on the basis of OD 280 nm and conductivity.

9. Freeze drying

The solution resulting from the buffer exchange stage is divided intoaliquots, e.g., of 10 mg in vials of 100 ml, and then frozen, e.g., for2 hours at −80° C. The frozen material is then lyophilized, e.g., usinga CT 60e (Heto) freeze-drier for 72 hours.

Alternative methods of protein isolation and purification can be usedand are well known to those of ordinary skill in the field of molecularbiology. Various protocols are described in Current Protocols inMolecular Biology, Chapter 10.

The new methods described herein can also be used to produce variousbiologically active fragments of the pro-UK flexible loop mutants. Forexample, one set of active fragments is known as low molecular weight(“LMW”) pro-UK flexible loop mutants. These mutants have the samesequence as the full-length pro-UK mutants, but are cleaved at theLys¹³⁵ amino acid location of the molecule, e.g., with plasmin ortrypsin, to form a smaller size (33K vs. 50K) protein molecule. TheseLMW pro-UK mutants have improved diffusion characteristics because oftheir smaller size. These LMW pro-UK mutants can also be activated toproduce LMW two-chain mutant UK, e.g., by passing the single-chain formover a column of Sepharose®-bound plasmin. One can also producefull-length, activated pro-UK flexible loop mutants by passing thepro-UK mutants over plasmin bound to Sepharose®, e.g., in columns or inbatch methods.

Storing and Administering Pro-UK Mutants

Once the pro-UK mutants are made, they can be lyophilized or stored inphysiologically acceptable excipients, such as organic acids, e.g.,acetic acid at a pH of about 5.4. The pro-UK mutants are quite stable insuch acids, and can actually be stored over time and then administereddirectly to patients in such an acid solution. The pro-UK mutantproteins can also be combined with other drugs to form compositions thatcan then be administered to a patient in one solution.

In general, bolus or “loading” doses of the pro-UK mutants will be inthe 20-40 mg range. The intravenous infusion dose of pro-UK flexibleloop mutants such as M5 is about 120-200 mg/hour (e.g., 100, 125, 150,or 175 mg/hour), whereas the intra-arterial infusion rate will be 50-100mg/hour (e.g., 60, 70, 80, or 90/mg/hour). Intra-arterial administrationof pro-UK mutants will also provide additional efficacy and safety inthe treatment of stroke patients.

The pro-UK in lyophilized form can be administered with a device thatcontains the pro-UK powder in one compartment, and in a secondcompartment contains a pre-measured amount of an excipient, such assterile saline, purified water, or some other physiologically acceptablecarrier in which the pro-UK powder can be reconstituted. The first andsecond compartments are connected by a wall such that the wall can bebroken by the user of the device just prior to injecting the pro-UKsolution. Thus, the device can be stored for long periods of time, andthen the pro-UK powder can be reconstituted as required without the needto measure the amount of the excipient.

In addition, the pro-UK can be packaged in specific aliquots and atspecific concentrations, e.g., in predetermined dosages ready foradministration, along with instructions to administer the pro-UK mutant,such as a flexible loop mutant, e.g., M5, to a person exhibitingsymptoms of stroke or symptoms of a heart attack. For example, a plasticIV bag containing M5 for infusion can be labeled for usepost-operatively, after angioplasty, or upon observing symptoms of aheart attack or stroke. In addition, a syringe for administering a bolusinjection of M5, e.g., for use by an EMT in an ambulance, can bepre-loaded with a bolus of 20, 25, 30, 35, or 40 mg of a pro-UK mutant,such as M5, and labeled for immediate use on a person exhibitingsymptoms of an apparent stroke or a heart attack.

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

The following materials were used in the examples.

Single-chain t-PA was obtained from Genentech (San Francisco, Calif.).UK was prepared by plasmin activation of pro-UK as previously described(Pannell et al., Blood, 69:22-26, 1987), and its concentration wasstandardized against the UK International reference standard (NIBSC,London, UK). Glu-plasminogen was prepared from DFP-treated human bankplasma. Fragment E-2 was prepared as previously described (Liu et al.,J. Clin. Invest., 88:2012-2017, 1991). D-dimer, Soluble fibrin(Desafib), and Lys-plasmin were obtained from American Diagnostica Inc.(Greenwich, Conn.). Fibrinogen (human) and synthetic chromogenicsubstrate for plasmin (S2251) and UK (S2444) were obtained from KabiPharmacia Inc. (Franklin, Ohio). Fibrinogen was radiolabeled with ¹²⁵Iusing Iodogen (Sigma, St. Louis, Mo.). PAI-1 is available from DupontMerck (Washington, D.C.)

Example 1 Preparation of M5

The gene for native pro-UK has been well characterized (Verde et al.,Proc. Nat. Acad. Sci., USA, 81:4727-4731, 1984) and its cDNA wasavailable from Dr. Paolo Sarmientos (Farmitalia, Milano, Italy). Thesite-directed mutant [Lys³⁰⁰→His, M5] of pro-UK was constructed andexpressed in E. coli as follows: The cDNA of M5 was obtained bysite-directed mutagenesis after subcloning the HindIII-BamHI restrictionfragment from pFC16 plasmid containing the full-length cDNA of pro-UK(Orini et al., Eur. J. Biochem., 195:691-697, 1991) into an M13 vector(mp18). The expression plasmid for the mutant was constructed byreinserting the mutated HindIII-BamHI fragment into pFC16 and introducedinto an E. coli type-B strain. M5 was purified from sonicated celllysates by chromatography through S-Sepharose®, pro-UK antibody affinitycolumn and Sephadex® G-25 after refolding by standard methods previouslydescribed (Winkler et al., Biochemistry, 25:4041-4045, 1986). Traceamounts of two-chain M5 (tcM5) were removed by passage over benzamidineSepharose® followed by treatment with DFP as previously described (Liuet al., J. Biol. Chem., 270:8408-8410, 1995). Purified M5 was observedas a single band on reduced SDS-PAGE. Protein concentration wasdetermined from absorbance at 280 nm using the extinction coefficient(E^(1%) _(280nm)=1.36) for pro-UK. Plasmin resistant mutations(Ala¹⁵⁸-proUK and Ala¹⁵⁸-M5) were also made by an additionalsite-directed mutagenesis at Lys¹⁵⁸ (Lys¹⁵⁸→Ala) in both pro-UK and M5.

Example 2 Enzymology of M5

Assay of Plasmin Sensitivity:

Since the activatability of pro-UK is essential for its fibrinolyticefficacy, this property had to be verified. A range of concentrations ofpro-UK or M5 (0-5 μmol/L) was incubated with Lys-plasmin (0.1 nmol/L) inthe presence of synthetic substrate (S2444, 1.2 mmol/L) in the assaybuffer (0.05 mol/L Tris HCl, 0.10 mol/L NaCl and 0.01% Tween80®, pH 7.4)at room temperature. The same range of concentrations of pro-UK or M5without plasmin was incubated with S2444 as control. The 0.1 n mol/Lplasmin had no direct effect on S2444 hydrolysis. The rate of pro-UK orM5 activation was calculated from the OD increase over time squared at410 nm on a microtiter plate as previously described (Liu et al., Blood,1993, 81:980-987). The kinetic constants were derived by Lineweaver-Burkanalysis.

Intrinsic Catalytic Activity Assay

For hydrolysis of S2444, pro-UK (1.0 μmol/L) or M5 (10.0 μmol/L) wasincubated with a range of concentrations (0-2.4 mmol/L) of S2444 in theassay buffer at room temperature. The reaction rate was measured by thelinear OD increase over time at 410 nm. 0.01-5.0 n mol/L of UKInternational Standards was used for the standard curve of S2444activity of UK. The kinetic constants were calculated fromLineweaver-Burk plots.

Activities of the two-chain (tc) derivatives of pro-UK and M5

1) Hydrolysis of S2444:

UK or tcM5 was prepared by plasmin treatment of the single chainprecursors as previously described (5). UK or tcM5 (4.0 nmol/L) wasincubated with a range of concentrations (0-2.4 mmol/L) of S2444 in theassay buffer at room temperature. The reaction rate was measured and thekinetic constants were calculated as described above.

2) Glu-Plasminogen Activation:

Time-absorbance curves of Glu-plasminogen activation were obtained bymeasuring the OD increase of the reaction mixture with time at theselected wavelength 410 nm. The reaction mixture contained S2251 (1.5 mmol/L), Glu-plasminogen (1.0-10.0 μmol/L) and UK or tcM5 (0.2 nmol/L).The reactants were made up in the assay buffer, and incubated at roomtemperature. The reaction rates were calculated from the OD increaseover time squared as previously described (19). The kinetic constantswere calculated from Lineweaver-Burk plots.

3) Glu-plasminogen activation by pro-UK or M5

Glu-plasminogen (2 μmol/L) was incubated with pro-UK or M5 (0.075 nmol/L) in the presence of 1.5 mmol/L S2251. The reaction rates werecompared from the OD increase over time.

The kinetic constants for M5 and pro-UK are shown in Table 4, whichshows that the catalytic efficiency of two-chain M5 is about twice thatof UK (against the synthetic substrate for UK). We have also found thatthe specific activity of M5 is about 100 K-200 K IU/mg, which issomewhat higher than UK. Nevertheless, the rate of clot lysis is twiceas high, as indicated in the differences in kinetics shown in Table 2TABLE 2 Kinetics analysis of S2444 amidolysis by pro-UK, single-chainM5, UK & tcM5 and glu-plasminogen activation by UK & tcM5 kcat (min-1)KM (μM) kcat/KM (min μM)-1 F S2444 (0.03-2.4 mmol/L) Amidolysis pro-UK0.32 ± 0.15 51 ± 9  0.00627 1 M5 0.065 ± 0.030 52 ± 12 0.00125 0.2 UK180 ± 130 78 ± 15 2.31 1 tcM5 350 ± 110 75 ± 18 4.67 2 Glu-Plasminogen(0.1-20.0 μmol/L) Activation UK 18.1 ± 0.6  11.4 ± 2.1  1.59 1 tcM5 9.2± 1.8 3.6 ± 1.5 2.56 1.6

Promotion of pro-UK or M5 Induced Plasminogen Activation by Co-Factors

Fibrin fragment E selectively and potently promotes Glu-plasminogenactivation by the intrinsic activity of pro-UK. Therefore, the promotingeffect of fragment E2, prepared as previously described, on M5 wasevaluated using plasmin-resistant (Lys¹⁵⁸) mutants of pro-UK and M5. TheOD increase over time in the reaction mixture at 410 nm was measuredusing standard techniques. The reaction mixture contained 1.5 m mol/LS2251, Glu-plasminogen (2.0 μmol/L), and 1.0 mol/L Ala¹⁵⁸-proUK orAla¹⁵⁸-M5 with or without 5.0 μmol/L fragment E2, in the assay buffer atroom temperature. The effects of other fibrin analogs such as fibrinogen(3 μmol/L), soluble fibrin monomer (SFM, 1 μmol/L), and D-dimer (1μmol/L), were also tested since they do not promote plasminogenactivation by pro-UK. The results are shown in Table 3 below, whichshows mean values from one experiment done in triplicate. TABLE 3Glu-Plasminogen (2 μmol/L) activation by plasmin resistant Ala¹⁵⁸- proUKor Ala¹⁵⁸-M5 (1 nmol/L) in the presence of co-factors Reaction Rate (asΔA₄₀₅ × 10⁶/min²) Co-factors A158-ProUK A158-M5 Buffer Control 4.40 0.90Fibrinogen (3 μmol/L) 1.20 0.19 SFM (1 μmol/L) 3.80 0.62 Fragment E (1μmol/L) 29.1 7.90 D-dimer (1 μmol/L) 4.30 1.84

Fibrin-fragment E, which selectively promotes plasminogen activation bypro-UK (Liu et al., Biochemistry, 1992, 31:6311-6317), had a similareffect on M5, whereas D-dimer and soluble fibrin or fibrinogen inducedlittle or no promotion. For this study, plasmin-resistant mutants(Lys¹⁵⁸→Ala) of pro-UK and M5 were used to prevent interference from theelaboration of the two-chain enzymes (Table 3).

Inhibition of UK or tcM5 by PAI-1

UK or tcM5 was incubated with PAI-1 in the assay buffer at equal molarconcentrations of enzyme and inhibitor (2.0-8.0 nmol/L). After differenttimes of incubation at 25° C., 80 μL of the UK/PAI-1 or tcM5/PAI-1reaction mixture was added to 20 μL S2444 (final concentration was 1.2mmol/L). The amount of uninhibited UK or tcM5 was determined from itsinitial rate of hydrolysis of S2444 as measured at 410 nm. Theconcentration of free PAI-1 was then calculated by difference. Thesecond-order rate constants were determined by linear regression of aplot of 1/[E] vs. time (where [E] is the concentration of UK or tcM5 attime [t]) as previously described (Hekman et al., Biochemistry,27:2911-2918, 1988).

The tcM5 was inhibited by PAI-1 with a K_(i) of 1.3±0.3×10⁷ M⁻¹ sec⁻¹,which was comparable to that of UK (1.7±0.4×10⁷ M⁻¹ sec⁻¹).

Comparative Reactions of the two TC-UK's with Plasma Protease Inhibitors

The active two-chain derivatives of both M5 (tc-M5) and wild-type pro-UK(tc-u-PA) were generated by incubation at 100 μg/ml with plasmin (1.2μg/ml) in Hepes buffered saline (pH 7.4 and with 1 mg/ml BSA added) for60 min at 37° C. These were added respectively to pooled human bloodbank plasma at 5 μg/ml and incubated at 37° C. At various time points,aliquots of these incubation mixtures were removed and the reactionstopped by dilution into Laemmli SDS sample buffer. These samples werelater subjected to Laemmli SDS PAG electrophoresis. After the resultantslab gels were washed in Triton (to remove the SDS) and then in buffer,they were laid onto underlays consisting of agarose, casein to opacify,and purified plasmin-free human plasminogen (20 μg/ml) (Lenich et al.,Blood, 90:3579-86, 1997). This construct was incubated at 37° C. todevelop the zymograms and photographed at times as appropriate. Inzymography, the presence of plasminogen activator activity in anelectrophoretic band is revealed by the local lysis of the casein in theunderlay. Inhibitor complexes are seen as the higher molecular weightbands of lysis.

The tcM5 (FIG. 13A) was observed to be more rapidly inhibited than wasthe wild type tc-u-PA (FIG. 13B). For example, about half of the free M5enzyme seems to have disappeared after 10 minutes, whereas most of thetcpro-UK is still present after 20 to 30 minutes. In addition, the C1Inhibitor complex is prominent with tcM5 (FIG. 13A), but is barely seenwith tc-uPA (FIG. 13B). These results are indicative of a greaterreactivity of the active site of M5 compared to tcpro-UK. This propertyof more efficient inhibition of the two-chain form, which will begenerated at the lysing thrombus surface, better confines plasminogenactivation to this site, thus further explaining the sparing of distanthemostatic fibrin.

The principal inhibitor complexes in the plasma were identified to be C1Inhibitor (also known as C1-Inactivator) and Antithrombin III with C1Inhibitor appearing to be the most avid. Beyond inferences from themolecular weight of the inhibitor complexes observed in zymograms, theidentity of complexes with C1 Inhibitor and with Anti-Thrombin III wereconfirmed by their comigration with complexes formed with commerciallyavailable purified inhibitors and by immuno-adsorption experiments usingcommercially available antibodies.

Example 3 Stability (Inertness) of M5 in Human Plasma Compared withPro-UK,

M5 (0-14 μg/mL) or pro-UK (0-3.0 μg/mL) was incubated (37° C.) in 1.0 mLof citrate pooled bank plasma. After six hours, 0.2 ml of a protinin(10,000 KIU/mL) was added and the fibrinogen remaining in the plasma wasmeasured by the thrombin-clottable protein method (Swaim et al., Clin.Chem., 13:1026-1028, 1967) and compared with the baseline value.

Under these conditions, M5 remained inert and did not induce fibrinogendegradation until its concentration exceeded 8 μg/mL (see FIG. 2F), andat 10 μg/mL, 30±6% fibrinogen remained. By contrast, pro-UK inducedfibrinogen degradation at a concentration greater than about 2 μg/mL(see FIG. 2C).

Example 4 In Vitro Clot Lysis by Pro-UK or M5 in Human Plasma

A previously standardized technique using radiolabeled plasma clotsincubated in plasma was used (Gurewich et al., J. Clin. Invest.,73:1731-1739, 1984). ^(I25)-labelled fibrinogen clots were prepared from0.2 mL plasma and incubated in 4 mL plasma. A range of fibrin specific(<25% fibrinogen loss) concentrations of pro-UK (0.5-3.0 μg/mL) or M5(0.5-14.0 μg/mL) was tested. Clot lysis was expressed as cpm of thelysis value against time. Fibrinogen was assayed (Swaim et al., Clin.Chem., 13:1026-1028, 1967) at the end of complete clot lysis or at sixhours, whichever came first.

As shown in FIG. 2D clot lysis in this plasma milieu with M5 remainedfibrin specific (<25% fibrinogen degradation) up to a concentration of8.0 μg/mL, whereas as shown in FIG. 2A, the upper limit for pro-UK was1.0 μg/mL. The maximum rate of clot lysis, determined from the slopes ofthe clot lysis curves, was about 40-50% per hour for pro-UK and about70-100% per hour for M5.

FIG. 2A shows that concentrations of 0.5 and 1.0 μg/mL of pro-UK werespecific, while concentrations of 1.5, 2.0, and 3.0 μg/mL were convertedinto UK, and thus were no longer specific. FIG. 2B shows theconcentration-dependent specificity cut-off nicely (based on plasminogenremaining) for pro-UK at about 1.0 μg/mL, whereas M5 was stable, andcaused no plasminogen degradation. FIG. 2C, shows the same cut-off forpro-UK based on fibrinogen remaining. Again, M5 was stable and did notdegrade fibrinogen.

When M5 (2 μg/mL) was combined with a small amount (30 ng/mL) of t-PA,insufficient to induce clot lysis by itself, the lag-phase was reducedby half. This has been ascribed to the creation of new (fragment E)plasminogen binding sites by t-PA-induced lysis, which promoteplasminogen activation by pro-UK and M5 (see Table 5). Therefore,fibrin-dependent plasminogen activation by M5 is similarly complementaryto t-PA (promoted by fragment D) as pro-UK (data not shown).

FIG. 2D shows that M5 caused specific clot lysis up to a concentrationof 8.0 μg/mL, whereas concentrations of 10, 12, and 14 μg/mL causednon-specific lysis because the M5 was converted into mutant UK. What issignificant, is that the rate of lysis by M5 at a concentration of 6 or8 μg/mL was essentially the same as the rate of lysis when it becausenon-specific. FIGS. 2E and 2F show the results of plasminogen remainingand fibrinogen remaining, respectively. Both graphs show that M5 remainsstable until a concentration of about 8.0 μg/mL.

Example 5 In Vivo Studies with M5

All procedures in animals were in accordance with the Guide for the Careand Use of Laboratory Animals (National Academy of Sciences, 1996) andwere approved by the Animal Studies Committee of Nanjing University.

Clot Lysis in Anesthetized Dogs

Male, mongrel dogs weighing 10-15 kg were anesthetized withpentobarbital sodium and maintained breathing room air. An experimentalmodel comparable to one previously used to evaluate the fibrinolyticproperties of pro-UK was used (Gurewich et al., 1984, supra). Clots wereformed from 1 mL native whole dog blood to which radiolabeled fibrinogen(1.9 μCi, 0.75 mCi/mg protein) and thrombin (10 units) were added. After20 minutes, a time when clot retraction had gone to completion, theclots were washed with saline three times and then cut into small (˜1mm³) pieces and injected through a 16-gauge needle into the femoralvein. After 15 minutes, a blood sample was obtained from a cannula inthe contralateral femoral vein for measurement of baselineradioactivity. Then an intravenous infusion of saline or activator wasstarted. Infusion rates of pro-UK (20 μg/kg/min) or t-PA (10 μg/kg/min),which have been reported in the literature to be both effective andfibrin-specific in dogs, were used. The t-PA infusion was limited to 60minutes due to its high cost. The other infusions were for 90 minutes.M5 was given at infusion rates of 20, 40, and 60 μg/kg/minutes. Atintervals during the infusions, blood samples were obtained formeasurement of radioactivity and fibrinogen.

In these experiments, clot lysis by M5 was dose-responsive. Due to itsfour-fold greater stability in plasma, a three-fold higher infusion rate(60 μg/kg/h) than pro-UK was possible with M5, similar to what was foundin the in vitro clot lysis experiments. As shown in the graph in FIG. 3,at this dose, M5 induced rapid lysis reaching 100% in <45 minutes. Lysiswith M5 was also more efficient, since the total quantity of activatorneeded to achieve 50% lysis was ˜600 μg/kg for M5 compared with 1200μg/kg for pro-UK. Higher infusion rates of pro-UK or t-PA were precludeddue to non-specific effects, which cause not only excessive bleeding,but also the “plasminogen steal” phenomenon, which can inhibit clotlysis. At lower doses (40 and 20 μg/kg/min), M5 induced comparable orless clot lysis as 20 μg/kg/minute of pro-UK or 10 μg/kg/minute of t-PA(possibly due to its longer lag phase). The results are summarized inFIG. 3. The number of dogs in each group is shown in parentheses. Lysisof lung clots in dogs by M5 (20, 40, 60 μg/Kg/min) vs t-PA and pro-UK.Lysis was dose-responsive by M5 and at the highest dosage, lysed theclots twice as quickly and twice as efficiently. The doses of pro-UK andt-PA chosen were those used in the literature and found to be optimal.

Plasma fibrinogen concentration in the dogs infused with the highestdose of M5 were 72%, 65% and 52% of the baseline value at 30, 45, and 60minutes respectively.

Assessment of Hemostasis in Dogs

In all of the dogs, bleeding from a standardized incision was measured.A 1 cm² skin incision was made over the shaved abdomen and the epidermispeeled off. One exposed superficial vessel was cut with a scalpel andthe bleeding site dabbed every 30 seconds with filter paper until bloodflow stopped. This was the primary bleeding time (BT), and was carriedout in adjacent 1 cm² wounds at 0, 20, and 60 minutes. Total bleedingwas also measured by counting the total number of standard (5×5 cm)gauze pads needed to absorb the blood oozing from the wounds. Each gauzepad was replaced after it was totally discolored by blood. Thismeasurement represented secondary bleeding since it came predominantlyfrom the two previous BT sites at which hemostasis had occurred. Thisprocedure was carried out over the first 60 minutes of each infusion.

The bar graph in FIG. 4 summarizes the results. The baseline primary BTin the 16 dogs was ˜1.2 minutes and this did not change significantlyduring the infusion in the four saline control dogs. At 20 minutes afterthe start of the infusion, the primary BT in the t-PA and pro-UK infuseddogs increased to ˜2.4 minutes, and after 60 minutes, the t-PA animalshad a primary BT>5 minutes compared with ˜4 minutes for pro-UK. Bycontrast, in the dogs infused with the maximum dose (60 μg/kg/min) ofM5, there was no increase at 20 minutes and the primary BT increasedinsignificantly to ˜1.5 minutes at 60 minutes. Mean values iSD areshown. The number of dogs in each group is shown in parentheses.

The total blood loss, which was measured by the number of blood-soakedgauze pads overlying the wounds, reflected secondary bleeding since thisblood loss came predominantly from rebleeding from the primary BT siteswhere hemostasis had occurred. As shown in the bar graph in FIG. 5, thisincreased more than eight-fold with t-PA, five-fold with pro-UK, but wasnot significantly increased by the maximum dose of M5. Mean values±SDare shown.

Clots Lysis and Hemostasis in Rhesus Monkeys

Rhesus monkeys represent a second species with a sensitivity to humanpro-UK/UK comparable to that of man, in contrast to most otherexperimental animals. Some modification of the experimental protocol wasnecessary to accommodate regulations pertaining to the experimental useof monkeys, which include a requirement that their life be preserved.

Six adult Rhesus monkeys (3 males & 3 females) weighing 5.8-8.6 kg wereanesthetized with intravenous sodium pentobarbital (30 mg/kg, I.V.). Apolyethylene catheter was placed into each brachial vein and used forblood collection and infusion respectively. A 2 mL sample of whole bloodwas mixed with radio-iodinated human fibrinogen (4.5×10⁶ cpm) andthrombin (20 units) in a plastic tube, and incubated at 37° C. for 20minutes. The whole blood clot was cut into ˜1 mm³ pieces and washed withsaline six times. The clots (containing 3.3×10⁶ cpm) were suspended in 5mL of saline and injected through the right brachial vein. After 30minutes, a blood sample from the contralateral brachial vein wasobtained for baseline radioactivity and then an infusion of saline (2monkeys) or M5 (4 monkeys) was started. M5 was given at the maximuminfusion rate used in the dogs (60 μg/kg/min) for 60 minutes. Atintervals during the infusion, blood samples were obtained formeasurement of radioactivity and fibrinogen. A BT was measured at 0, 30,45, and 60 minutes from a cut over the lower abdomen that was 5 mm inlength and 1 mm in depth using a sterile lancet. The BT was performed bythe standard method using filter paper dabbing the cut every 30 secondsuntil bleeding stopped. Rebleeding from the BT sites where hemostasishad taken place at the earlier time points was evaluated.

As shown in the bar graph of FIG. 6, M5 infused at 60 μg/kg/minuteinduced 100% clot lysis within 60 minutes in all four monkeys comparedwith 8% (not shown) in the two saline infused animals. The fibrinogenconcentrations at 30, 45, and 60 minutes of the infusion were 78%, 66%,and 57% of the baseline values respectively, similar to the responseobserved in the dogs and consistent with the observation that these twospecies have a comparable sensitivity to human pro-UK/UK. Mean±SD valuesexpressed as a percent of baseline are shown.

FIG. 6 also shows the primary BT (252±2 sec), expressed as a percent ofbaseline (100%), at 30 minutes was reduced to 85% (215±18 sec) returningto baseline at 45 minutes, and increasing insignificantly to 108%(272±25 sec) at 60 minutes. Mean±SD values are shown. The BT in the twosaline controls followed a similar pattern (not shown). Rebleeding fromthe BT sites at which primary hemostasis had occurred was not seenduring the M5 infusions, consistent with the dogs infused with M5.

Clot Lysis of an Arterial Thrombus in Dogs

In this dog study, we studied rates of lysis of a femoral arterythrombus using the Badylak model (Badylak et al., J. PharmacologicalMethods, 1988, 19:293-304). In brief, ten dogs (8-10 Kg) wereanesthetized with sodium pentobarbital, a 2 cm segment of the leftfemoral artery was isolated, injury was induced by the infusion of hot(100° C.) water into the segment, and then the segment was filled withdog whole blood containing radiolabeled fibrinogen. After allowing time(30 minutes) for clot attachment, the ligatures were released and theintravenous infusions begun. T-PA was infused at a rate of 10 μg/Kg/min.and M5 was infused at a rate of 60 μg/Kg/min. Both infusions were for 90minutes. Lysis was measured by the reduction in radioactivity measuredby a probe fixed above the femoral artery thrombus, and flow rates weremeasured with a flow meter.

The infusion rate of t-PA used herein was the one shown in theliterature to give optimal lysis (Young et al., Thrombosis andHaemostasis, 1995, 74:1348-1352). At higher infusion rates of t-PA,non-specific effects begin to predominate, which are associated withplasminogen consumption (“plasminogen steal” phenomenon), which impairslysis due to the loss of substrate.

The mean and standard deviations for lysis and reflow are shown in FIGS.14A and 14B. As shown in FIG. 14A, M5 achieved 30% clot lysis withinabout 13 minutes, whereas it took tPA over 45 minutes to reach the samelevel. Both M5 and tPA reached 100% clot lysis after about 85 minutes.As shown in FIG. 14B, the reflow rate was more rapid in the M5-infuseddogs compared with the t-PA-infused dogs. In FIG. 14B, “linear M5” and“liner t-PA” refers to their mean flow rates. These results indicatethat M5 provided significantly faster lysis and reflow than did t-PA.

Summary of In Vivo Results

A rapid lysis of clots by M5 was observed in dogs. In parallel with thein vitro findings, M5 was less efficient than pro-UK or (t-PA) atcomparable doses, but at a three-fold higher infusion rate, M5 induced100% clot lysis within 45 minutes compared with 30% or less by the othertwo activators. The rapidity of lysis by M5 made it also more efficient,since the total amount of activator needed to induce lysis of 50% theclots was only 600 μg/kg for M5 compared with ˜1200 μg/kg for pro-UK(FIG. 3). Higher infusion rates with pro-UK were precluded by its moreready conversion to UK, resulting in non-specific effects. Since thelysis properties of M5 in vitro and in vivo were comparable and reflectthe catalytic changes induced by the mutation, it reasonably expectedthat these properties will also be seen upon administration to humans.

Some fibrinogen degradation was observed at the highest dose of M5,reflecting non-specific plasminogen activation. However, this wasinsufficient to interfere with hemostasis, since no significant increasein either the primary bleeding time (FIG. 4) or total blood loss (FIG.5) occurred. The latter was predominantly related to rebleeding at theprimary BT sites and therefore corresponds to secondary bleeding,suggesting that hemostatic fibrin was spared by M5. By contrast, theprimary BT and secondary bleeding increased four to eight-fold in thepro-UK and t-PA treated dogs, whereas clot lysis was at least two-foldless effective in these animals. It is noteworthy that bleeding has notbeen well correlated with non-specific plasminogen activation and thatsome highly fibrin-specific activators interfere significantly withhemostasis (Montoney et al., Circulation, 91:1540-1544, 1995).

It may be postulated that bleeding during fibrinolysis reflects avulnerability of hemostatic fibrin to the activator. The most bleedingoccurred with t-PA, which is consistent with the paradoxically higherrate of intracranial bleeding associated with this activator. Theinfusion rate at which intravascular clot lysis by M5 was the most rapidinduced little or no rebleeding at the BT sites where hemostasis hadoccurred. Therefore, this hemostatic fibrin in dogs and monkeys appearedto be resistant to the thrombolytic properties of M5. These findingsattest to the presence of certain apparent differences between fibrin ina thrombus and hemostatic fibrin. Some differences may arise from thefact that only a thrombus occludes a vessel. Stasis can trigger thelocal release of t-PA from the endothelium and facilitate its binding tothe thrombus, initiating some fibrin degradation. As a result, newplasminogen binding sites are exposed. Plasminogen bound to these newsites (fibrin fragment E) is selectively activated by pro-UK, a propertyretained by M5. By contrast, fibrin fragment D (intact fibrin) promotesplasminogen activation by t-PA. This difference may help explain thelower BT and blood loss by pro-UK than t-PA. Since M5 is more stable inblood than pro-UK, this selective fibrinolytic mechanism is betterpreserved at pharmacological doses.

Example 6 Acute Myocardial Infarction (AMI)

A patient arrives at the Emergency Room (ER) of a hospital with symptomsof AMI. A loading dose (20-40 mg) of M5 is immediately injected (due toits safety, delay in treatment until triage and diagnosis is confirmedis not necessary). Thereafter, an EKG is taken and blood tests areobtained to validate the diagnosis. If AMI is confirmed, an infusion ofM5 (100-120 mg/h) is started immediately. Then a catheterization team isassembled, the angioplasty room is prepared, and the patient istransported there for this procedure. Since experience shows that thistakes' 60-90 minutes, thrombolysis will have been completed by the timethe angiogram is taken. If significant stenosis due to atheromatousplaque is present, angioplasty and stent placement is performed, butthis may be unnecessary. Thus M5 leads to more accurate diagnosis andproper treatment.

This early treatment is especially important in diabetic patients,because the diabetic myocardium tolerates ischemia significantly lesswell than the non-diabetic, making early reperfusion critical. About 30%of AMI is in diabetics and is associated with a significantly highermortality. In addition, thrombolytics are rarely given to patients overthe age of 75 because of higher rates of hemorrhagic complications inthis age group. At the same time, the mortality from AMI is much higherin the age group and the therapeutic benefit of therapeutic thrombolysisis correspondingly higher. Thus, pro-UK flexible loop mutants such as M5are especially beneficial in diabetics and older patients.

Example 7 Angioplasty/Stent Procedures

A patient arrives at a hospital with new onset angina pectoris, unstableangina, or exacerbation of existing angina. All of these clinicalscenarios are consistent with ischemia due to new compromise ofperfusion in one of the coronary arteries, which is invariably relatedto some clotting on an athereromatous plaque.

A bolus of M5 (as in Example 6) is given and an infusion is started.After further evaluation (triage) an interventional procedure iselected. Preparations for an elective angioplasty/stent are then made.Since it has been shown that removal of thrombus improves the outcome ofthese procedures, this pretreatment with M5, which is not possible withexisting thrombolytics, represents a significant improvement overcurrent management of these patients.

Example 8 Stroke

A patient with arterial fibrillation arrives at the ER 3 hours after thesudden onset of hemiplegia. A bolus of M5 and infusion (as in Example 6)are immediately started.

A CT scan of the head is performed showing ischemia, but no hemorrhage.Current evidence indicates that when reperfusion is achieved within 6hours of a stroke, significant recovery of brain function is achieved.This is currently not possible because the benefits of t-PA (the onlydrug approved by FDA for the U.S.) are limited to 3 hours.

Example 9 Post-Operative Usage

A post-operative patient sustains a major pulmonary embolus (a notuncommon post-operative complication). M5 is administered by infusionand/or bolus at the dosages indicated in Example 6.

Note, in the post-operative period (2-3 weeks) all currently availablethrombolytic drugs have been strictly contraindicated due to anextremely high risk of major hemorrhage, related to lysis of hemostaticfibrin in the surgical wound. Therefore, this patient would be deniedthrombolytic treatment, which in the case of a major embolus would belife-saving. Use of M5, on the other hand, spares hemostatic fibrin, andmay be used.

Example 10 Peripheral Artery Disease

A patient enters a hospital with worsening of his intermittentclaudication (due to peripheral artery disease). Although, with thesepatients, arterial disease is largely atherosclerotic, there is often asignificant clot overlay, which is susceptible to lysis. These patientshave not been given the benefit of currently available thrombolyticdrugs because the available drugs have been considered too toxic becauseof the bleeding effects. The safety of M5 can open up this indication,as it can be tried with little or no risk. If it does not work, surgerycan always be a second resort. Therefore M5 can be administered as inExample 6.

Example 11 Refractory Angina Pectoris

A patient with refractory angina pectoris is treated with a dosage of M5(150 mg) infused over one hour, 3 times per week for 12 weeks.

Example 12 Clearing of Dialysis Catheter

In all patients on chronic dialysis, blocked catheters due toaccumulation of fibrin material is a recurrent problem. To deal withthis problem, 5000 units of two-chain M5 (0.03 mg) are instilled toclear each port.

The present FDA approved labeling is for 0.05 mg UK (Abbokinase®), butwhen this was taken off the market, it was replaced by 1 mg of t-PA.Abbokinase® was recently reintroduced to the market, but it is lesspotent than mutant UK, such as tcM5. Abbokinase® is also a low moleculeweight form, which is a less efficient plasminogen activator than highmolecular weight UK. The use of low molecular weight mutant UK (e.g.,LMW tcM5) would significantly improve results compared to the use ofdialysis catheters, the dose is 5,000 IU per port. This is equivalent to0.025-0.05 mg and this induces the same hemodialysis blood flow rate as1 mg of t-PA.

Example 13 Preparation of M5

Competent cells of strain BL21/DE3 RIL were prepared using a calciumchloride procedure of Mandel and Higa (Mol. Biol., 53:154, 1970). 200 μlof a preparation of these cells at 1×10⁹ cells per milliliter wastransformed with 2 μl of plasmid pET29aUKM5 that encodes M5 (approximateconcentration 5 μg/ml). Transformants were selected on plates of L-agarcontaining 30 μg/ml kanamycin.

Plasmid pET29aUKM5 was made starting with plasmid pET29a (Novagen) shownin FIG. 9. As shown in FIGS. 10 and 11, to construct pET29-u-UKM5 forthe expression of pro-UK (M5), the pro-UK (M5) gene was amplified fromplasmid pFC16 by the polymerase chain reaction using the followingprimers: Primer 1 (5′ GAG GAT TAC ATA TGA GCA ATG AGC (SEQ ID NO:1) 3′),Primer 2 (5′ CTG GGG ACC GAG CTC TCA GAG, GGC (SEQ ID NO:2) CAG GCC ATT3′) Primer 3 (5′ GGC TTT GGA CAC GAG AAT TCT ACC (SEQ ID NO:3) GAC TATCTC 3′) Primer 4 (5′ AGA ATT CTC GTG TCC AAA GCC AGT (SEQ ID NO:4) GATCTC AC 3′)

Primer 3 and Primer 4 were used to mutate Lys³⁰⁰→His. Primer 1 andPrimer 2 were used to incorporate NdeI and SacI restriction sitesimmediately 5′ to the first codon and immediately 3′ to the stop codonof pro-UK cDNA, respectively. The amplified pro-UK M5 (M5) gene wasdigested with NdeI and SacI, purified, and ligated with the largefragment of NdeI-SacI-digested pET29a. The sequence of the M5 codingregion was confirmed by DNA sequencing.

Two small colonies were streaked with wooden toothpicks (each as threestreaks about 1 cm long) onto L-agar containing the same antibiotic.After 12 hours of incubation at 37° C., portions of the streaks weretested for M5 production by inoculation onto 10 ml of LB medium(containing kanamycin at a concentration of 30 μg/ml) and incubatedovernight at 37° C. The following day, the cultures were diluted 1:100in M9 medium, containing the same concentration of kanamycin, andincubated for 6 hours at 37° C. Total cell proteins from 250 μl aliquotsof culture (O.D.₅₅₀=1 to 1.5) were analyzed by sodium dodecylsulfatepolyacrylamide gel electrophoresis (SDS-PAGE) as described in Laemmli,Nature, 227:680, 1970. A major protein band having a molecular weightcorresponding to that of non-glycosylated M5 (45,000 daltons) wasobserved for the two independent samples.

FIG. 12 shows the SDS-PAGE results. Lane 1 includes molecular weightstandards. Lanes 2, 3, and 4 show the results of the supernatants at anOD of 1.5. Since M5 is an insoluble protein, we did not expect to findM5 in the supernatant, and found none. The band at about 45 kDa is asoluble host protein. Lane 2 contains BL21(DE3)RIL[pET29a], which lacksthe M5 encoding gene. Lane 3 contains BL21(DE3)RIL[M5-PUK], which hasthe M5 encoding gene, and the RIL strain, but since M5 is insoluble, wefound none in this lane. Lane 4 BL21(DE3) [M5-PUK], which has the M5encoding gene, but not the RIL strain. Again, there was no M5 in thesupernatant.

Lanes 5, 6, and 7 show the results of the pellet (“inclusion bodies”),again at an OD of 1.5. Lane 5 contains BL21(DE3)RIL[pET29a] (no M5encoding gene), and as expected, it shows no M5. Lane 6 containsBL21(DE3)RIL[M5-PUK], which has the M5 encoding gene, and the RILstrain, and shows a very high level of M5. Lane 7 contains BL21(DE3)[M5-PUK], which has the M5 encoding gene, but not the RIL strain. Thisstrain does produce some M5, but not nearly as much as the RIL strain.

A set of streaks corresponding to colony no. 2 (clone 2) was chosenarbitrarily for further characterization and then selected as an M5producing strain.

Example 14 Testing of Alternate Host Cells

Using the procedure described herein and in Example 13, severaladditional E. coli host strains were screened with the objective toisolate a transformant strain able to produce M5 at high levels. PlasmidpET29aUKM5 in the following strains: BL21/DE3, BL21/DE3 pLys, JM109/DE3,and HB 101/DE3. None of these strains, when transformed with plasmidpET29aUKM5, was able to yield high quantities of the M5 polypeptide asseen with the host strain BL21/DE3 RIL, indicating that indeed thecombination of the specific expression plasmid with strain BL21/DE3 RILis an important combination to obtain high quantities of M5. Forexample, the following yields were obtained: Host strain ProductivityBL21(DE3) RIL 4.12 grams/liter BL21(DE3) 0.91 grams/liter BL21(DE3)pLys0.82 grams/liter

The productivity is expressed as quantity of M5 polypeptide as measuredby SDS PAGE analysis and quantified against a standard of pro-UK. Thus,it is clear that the combination of the specific plasmid and its PhageT7 promoter sequences and strain BL21/DE3/RIL provides a far greateryield than even other type B strains. This data quantifies the resultsseen in Lanes 6 and 7 of FIG. 12. Although the E. coli type B strainBL21 (DE3) produces some M5, the BL21(DE3)RIL strain produces about 4.5times as much M5.

Example 15 Fermentation of the Host Cells

Transformed bacterial cells are cultured at high biomass in appropriatefermentors as follows. A first fermentation phase was carried out inErlenmeyer flasks to obtain a seed culture large enough to inoculate theproduction stage (second fermentation stage). One vial of working cellbank (0.1 ml) was diluted in 100 ml of sterile EC1 medium (details areprovided in Table 4 below) and grown at 37° C. overnight with theagitation of 220 rpm.

The working cell bank was made of a glycerol suspension of an overnightLB culture containing kanamycin at 30 μg/ml and chloramphenicol at 30μg/ml of the M5 producing strain (BL21/DE3 RIL carrying plasmidpET29aUKM5). TABLE 4 Medium EC-1 Per liter: Glucose 10 g Yeast extract 1g (NH₄)2HPO₄ 2 g K₂HPO₄ 6.75 g MgSO₄ × 7H₂O 0.7 g Citric acid 0.85 g TMS(see below) 5 ml TMS: Per liter of 5M HCl FeSO₄ × 7 H₂O 10 g ZnSO₄ × 7H₂O 2.25 g CaCl₂ × 2 H₂O 2 g CuSO₄ × 5 H₂O 1 g MnSO₄ × 5 H₂O 0.23 gNa2B₄O₇ × 10 H₂O 0.23 g (NH₄)₆MO₇O₂₄ 0.1 g

A second fermentation stage includes the following steps:

(1) 20 ml of the seed culture, prepared in the Erlenmeyer flask, wasadded to 2.0 liters of EC-1 medium in a fermentor;

(2) the pH in the fermentor was kept at 6.8 using a solution of 28%(v/v) ammonia water;

3) D.O. was maintained at 40% of air saturation by increasing theagitation speed and by changing the percentage of pure oxygen;

(4) the temperature of fermentation was kept at 35° C.; and

(5) a nutrient feeding solution (described in further detail in Table 5below) was added exponentially when all the glucose initially present isconsumed (usually after 8 hours), following the equation V=Vo e^(0.18t),where V=volume of feeding solution added (ml/h), Vo= 1/100 of thestarting fermentation medium (ml), and t=time of fermentation after thestart of the feeding phase (hours). TABLE 5 Feeding Solution Per liter:Glucose 400 g Yeast extract 100 g

In this method, gene expression was induced by adding IPTG at a finalconcentration of 1.2 mM, when the fermentation reached a cell density of90 OD₆₀₀.

The post-induction fermentation was prolonged for 6 hours to allow thecells to produce M5. Samples of 0.5 ml were removed every 2 hours foranalysis.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A method of treating a patient with symptoms of a heart attackcomprising: determining that the patient potentially has had a heartattack based on observing one or more symptoms of a heart attack; andadministering to the patient a composition comprising an amount of apro-urokinase mutant polypeptide (pro-UK mutant) effective to lyse anypotential blood clot causing the symptoms of a heart attack.
 2. Themethod of claim 1, wherein the composition is administered within 24hours of the onset of symptoms.
 3. The method of claim 1, wherein thecomposition is administered within two hours of the onset of symptoms.4. The method of claim 1, wherein the composition is administered byintravenous infusion at a pro-UK mutant dosage of 100-200 mg/hour. 5.The method of claim 1, further comprising: obtaining a medicalconfirmation of acute myocardial infarction; and administering aninfusion of the composition at a pro-UK mutant dosage of 100-120 mg/hourfor 60 to 90 minutes.
 6. The method of claim 1, wherein the compositionis administered prior to angioplasty.
 7. The method of claim 1, whereinthe composition is administered as a bolus comprising 20-50 mg of thepro-UK mutant.
 8. The method of claim 1, wherein the composition isadministered as a bolus and an infusion and wherein the total dose ofthe bolus and infusion together is 20-50 mg.
 9. The method of claim 1,wherein the pro-UK mutant is a flexible loop mutant.
 10. The method ofclaim 9, wherein the flexible loop mutant has the amino acid Lysinereplaced by the amino acid Histidine at position 300.